Methods of making modified viral genomes

Abstract

This invention provides an attenuated virus which comprises a modified viral genome containing nucleotide substitutions engineered in multiple locations in the genome, wherein the substitutions introduce synonymous deoptimized codons into the genome. The instant attenuated virus may be used in a vaccine composition for inducing a protective immune response in a subject. The invention also provides a method of synthesizing the instant attenuated virus. Further, this invention further provides a method for preventing a subject from becoming afflicted with a virus-associated disease comprising administering to the subject a prophylactically effective dose of a vaccine composition comprising the instant attenuated virus.

Description

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FIELD OF THE INVENTION

The present invention relates to the creation of an attenuated virus comprising a modified viral genome containing a plurality of nucleotide substitutions. The nucleotide substitutions result in the exchange of codons for other synonymous codons and/or codon rearrangement and variation of codon pair bias.

BACKGROUND OF THE INVENTION

Rapid improvements in DNA synthesis technology promise to revolutionize traditional methods employed in virology. One of the approaches traditionally used to eliminate the functions of different regions of the viral genome makes extensive but laborious use of site-directed mutagenesis to explore the impact of small sequence variations in the genomes of virus strains. However, viral genomes, especially of RNA viruses, are relatively short, often less than 10,000 bases long, making them amenable to whole genome synthesis using currently available technology. Recently developed microfluidic chip-based technologies can perform de novo synthesis of new genomes designed to specification for only a few hundred dollars each. This permits the generation of entirely novel coding sequences or the modulation of existing sequences to a degree practically impossible with traditional cloning methods.

Such freedom of design provides tremendous power to perform large-scale redesign of DNA/RNA coding sequences to: (1) study the impact of changes in parameters such as codon bias, codon-pair bias, and RNA secondary structure on viral translation and replication efficiency; (2) perform efficient full genome scans for unknown regulatory elements and other signals necessary for successful viral reproduction; and (3) develop new biotechnologies for genetic engineering of viral strains and design of anti-viral vaccines.

As a result of the degeneracy of the genetic code, all but two amino acids in the protein coding sequence can be encoded by more than one codon. The frequencies with which such synonymous codons are used are unequal and have coevolved with the cell's translation machinery to avoid excessive use of suboptimal codons that often correspond to rare or otherwise disadvantaged tRNAs (Gustafsson et al., 2004). This results in a phenomenon termed “synonymous codon bias,” which varies greatly between evolutionarily distant species and possibly even between different tissues in the same species (Plotkin et al., 2004).

Codon optimization by recombinant methods (that is, to bring a gene's synonymous codon use into correspondence with the host cell's codon bias) has been widely used to improve cross-species expression (see, e.g., Gustafsson et al., 2004). Though the opposite objective of reducing expression by intentional introduction of suboptimal synonymous codons has not been extensively investigated, isolated reports indicate that replacement of natural codons by rare codons can reduce the level of gene expression in different organisms. See, e.g., Robinson et al., 1984; Hoekema et al., 1987; Carlini and Stephan, 2003; Zhou et al., 1999. Accordingly, the introduction of deoptimized synonymous codons into a viral genome may adversely affect protein translation and thereby provide a method for producing attenuated viruses that would be useful for making vaccines against viral diseases.

Viral Disease and Vaccines

Viruses have always been one of the main causes of death and disease in man. Unlike bacterial diseases, viral diseases are not susceptible to antibiotics and are thus difficult to treat. Accordingly, vaccination has been humankind's main and most robust defense against viruses. Today, some of the oldest and most serious viral diseases such as smallpox and poliomyelitis (polio) have been eradicated (or nearly so) by world-wide programs of immunization. However, many other old viruses such as rhinovirus and influenza virus are poorly controlled, and still create substantial problems, though these problems vary from year to year and country to country. In addition, new viruses, such as Human Immunodeficiency Virus (HIV) and Severe Acute Respiratory Syndrome (SARS) virus, regularly appear in human populations and often cause deadly pandemics. There is also potential for lethal man-made or man-altered viruses for intentional introduction as a means of warfare or terrorism.

Effective manufacture of vaccines remains an unpredictable undertaking. There are three major kinds of vaccines: subunit vaccines, inactivated (killed) vaccines, and attenuated live vaccines. For a subunit vaccine, one or several proteins from the virus (e.g., a capsid protein made using recombinant DNA technology) are used as the vaccine. Subunit vaccines produced in Escherichia coli or yeast are very safe and pose no threat of viral disease. Their efficacy, however, can be low because not all of the immunogenic viral proteins are present, and those that are present may not exist in their native conformations.

Inactivated (killed) vaccines are made by growing more-or-less wild type (wt) virus and then inactivating it, for instance, with formaldehyde (as in the Salk polio vaccine). A great deal of experimentation is required to find an inactivation treatment that kills all of the virus and yet does not damage the immunogenicity of the particle. In addition, residual safety issues remain in that the facility for growing the virus may allow virulent virus to escape or the inactivation may fail.

An attenuated live vaccine comprises a virus that has been subjected to mutations rendering it less virulent and usable for immunization. Live, attenuated viruses have many advantages as vaccines: they are often easy, fast, and cheap to manufacture; they are often easy to administer (the Sabin polio vaccine, for instance, was administered orally on sugar cubes); and sometimes the residual growth of the attenuated virus allows “herd” immunization (immunization of people in close contact with the primary patient). These advantages are particularly important in an emergency, when a vaccine is rapidly needed. The major drawback of an attenuated vaccine is that it has some significant frequency of reversion to wt virulence. For this reason, the Sabin vaccine is no longer used in the United States.

Accordingly, there remains a need for a systematic approach to generating attenuated live viruses that have practically no possibility of reversion and thus provide a fast, efficient, and safe method of manufacturing a vaccine. The present invention fulfills this need by providing a systematic approach, Synthetic Attenuated Virus Engineering (SAVE), for generating attenuated live viruses that have essentially no possibility of reversion because they contain hundreds or thousands of small defects. This method is broadly applicable to a wide range of viruses and provides an effective approach for producing a wide variety of anti-viral vaccines.

SUMMARY OF THE INVENTION

The present invention provides an attenuated virus which comprises a modified viral genome containing nucleotide substitutions engineered in multiple locations in the genome, wherein the substitutions introduce a plurality of synonymous codons into the genome. This substitution of synonymous codons alters various parameters, including codon bias, codon pair bias, density of deoptimized codons and deoptimized codon pairs, RNA secondary structure, CpG dinucleotide content, C+G content, translation frameshift sites, translation pause sites, the presence or absence of tissue specific microRNA recognition sequences, or any combination thereof, in the genome. Because of the large number of defects involved, the attenuated virus of the invention provides a means of producing stably attenuated, live vaccines against a wide variety of viral diseases.

In one embodiment, an attenuated virus is provided which comprises a nucleic acid sequence encoding a viral protein or a portion thereof that is identical to the corresponding sequence of a parent virus, wherein the nucleotide sequence of the attenuated virus contains the codons of a parent sequence from which it is derived, and wherein the nucleotide sequence is less than 90% identical to the nucleotide sequence of the parent virus. In another embodiment, the nucleotide sequence is less that 80% identical to the sequence of the parent virus. The substituted nucleotide sequence which provides for attenuation is at least 100 nucleotides in length, or at least 250 nucleotides in length, or at least 500 nucleotides in length, or at least 1000 nucleotides in length. The codon pair bias of the attenuated sequence is less than the codon pair bias of the parent virus, and is reduced by at least about 0.05, or at least about 0.1, or at least about 0.2.

The virus to be attenuated can be an animal or plant virus. In certain embodiments, the virus is a human virus. In another embodiment, the virus infects multiple species. Particular embodiments include, but are not limited to, poliovirus, influenza virus, Dengue virus, HIV, rotavirus, and SARS.

This invention also provides a vaccine composition for inducing a protective immune response in a subject comprising the instant attenuated virus and a pharmaceutically acceptable carrier. The invention further provides a modified host cell line specially engineered to be permissive for an attenuated virus that is inviable in a wild type host cell.

In addition, the subject invention provides a method of synthesizing the instant attenuated virus comprising (a) identifying codons in multiple locations within at least one non-regulatory portion of the viral genome, which codons can be replaced by synonymous codons; (b) selecting a synonymous codon to be substituted for each of the identified codons; and (c) substituting a synonymous codon for each of the identified codons.

Moreover, the subject invention provides a method of synthesizing the instant attenuated virus comprising changing the order, within the coding region, of existing codons encoding the same amino acid in order to modulate codon pair bias.

Even further, the subject invention provides a method of synthesizing the instant attenuated virus that combines the previous two methods.

According to the invention, attenuated virus particles are made by transfecting viral genomes into host cells, whereby attenuated virus particles are produced. The invention further provides pharmaceutical compositions comprising attenuated virus which are suitable for immunization.

This invention further provides methods for eliciting a protective immune response in a subject, for preventing a subject from becoming afflicted with a virus-associated disease, and for delaying the onset, or slowing the rate of progression, of a virus-associated disease in a virus-infected subject, comprising administering to the subject a prophylactically or therapeutically effective dose of the instant vaccine composition.

The present invention further provides an attenuated virus which comprises a modified viral genome containing nucleotide substitutions engineered in multiple locations in the genome, wherein the substitutions introduce a plurality of synonymous codons into the genome, wherein the nucleotide substitutions are selected by a process comprising the steps of initially creating a coding sequence by randomly assigning synonymous codons in respective amino acid allowed positions, calculating a codon pair score of the coding sequence randomly selecting and exchanging either (a) pairs of codons encoding the same amino acids or (b) substituting synonymous codons in accordance with a simulated annealing optimization function and repeating the previous step until no further improvement (no change in pair score or bias) is observed for a specific or sufficient number of iterations, until the solution converges on an optima or near optimal value

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Codon use statistics in synthetic P1 capsid designs. PV-SD maintains nearly identical codon frequencies compared to wt, while maximizing codon positional changes within the sequence. In PV-AB capsids, the use of nonpreferred codons was maximized. The lengths of the bars and the numbers behind each bar indicate the occurrence of each codon in the sequence. As a reference, the normal human synonymous codon frequencies (“Freq.” expressed as a percentage) for each amino acid are given in the third column.

FIGS. 2A-B. Sequence alignment of PV(M), PV-AB and PV-SD capsid coding regions. The nucleotide sequences of PV(M) (SEQ ID NO:1), PV-AB (SEQ ID NO:2) and PV-SD (SEQ ID NO:3) were aligned using the MultAlin online software tool (Corpet, 1988). Numbers above the sequence refer to the position within the capsid sequence. (FIG. 2A) Nucleotide 1 to nucleotide 1300; (FIG. 2B) nucleotide 1301 to nucleotide 2643. Nucleotide 1 corresponds to nucleotide 743 in the PV(M) virus genome. In the consensus sequence, the occurrence of the same nucleotide in all three sequences is indicated by an upper case letter; the occurrence of the same nucleotide in two of the three sequences is indicated by a lower case letter; and the occurrence of three different nucleotides in the three sequences is indicated by a period.

FIGS. 3A-J. Codon-deoptimized virus phenotypes. (FIG. 3A) Overview of virus constructs used in this study. (FIG. 3B) One-step growth kinetics in HeLa cell monolayers. (FIGS. 3C to H) Plaque phenotypes of codon-deoptimized viruses after 48 h (FIGS. 3C to F) or 72 h (FIGS. 3G and H) of incubation; stained with anti-3D^pol antibody to visualize infected cells. (FIG. 3C) PV(M), (FIG. 3D) PV-SD, (FIG. 3E) PV-AB, (FIG. 3F) PV-AB^755-1513, (FIGS. 3G and H) PV-AB^2470-2954. Cleared plaque areas are outlined by a rim of infected cells (FIGS. 3C and D). (FIG. 3H) No plaques are apparent with PV-AB^2470-2954 after subsequent crystal violet staining of the well shown in panel FIG. 3G. (FIGS. 3I and J) Microphotographs of the edge of an immunostained plaque produced by PV(M) (FIG. 3I) or an infected focus produced by PV-AB^2470-2954 (FIG. 3J) after 48 h of infection.

FIGS. 4A-E. Codon deoptimization leads to a reduction of specific infectivity. (FIG. 4A) Agarose gel electrophoresis of virion genomic RNA isolated from purified virus particles of PV(M) (lane 1), PV-AB^755-1513 (lane 2), and PV-AB^2470-2954 (lane 3). (FIG. 4B) Silver-stained SDS-PAGE protein gel of purified PV(M) (lane 1), PV-AB^755-1513 (lane 2), and PV-AB^2470-2954 (lane 3) virus particles. The three larger of the four capsid proteins (VP1, VP2, and VP3) are shown, demonstrating the purity and relative amounts of virus preparations. (FIG. 4C) Development of a virus capture ELISA using a poliovirus receptor-alkaline phosphatase (CD155-AP) fusion protein probe. Virus-specific antibodies were used to coat ELISA plates, and samples containing an unknown virus concentration were applied followed by detection with CD155-AP. Virus concentrations were calculated using a standard curve prepared in parallel with known amounts of purified wt virus (FIG. 4E). (FIG. 4D) The amounts of purified virus and extracted virion RNA were spectrophotometrically quantified, and the number of particles or genome equivalents (1 genome=1 virion) was calculated. In addition, virion concentrations were determined by ELISA. The infectious titer of each virus was determined by plaque/infected-focus assay, and the specific infectivity was calculated as PFU/particle or FFU/particle.

FIGS. 5A-B. In vitro translation of codon-deoptimized and wild type viruses. The PV-AB phenotype is determined at the level of genome translation. (FIG. 5A) A standard in vitro translation in HeLa S10 extract, in the presence of exogenously added amino acids and tRNAs reveals no differences in translation capacities of codon-deoptimized genomes compared to the PV(M) wt. Shown is an autoradiograph of [³⁵S]methionine-labeled translation products resolved on a 12.5% SDS-PAGE gel. The identity of an aberrant band (*) is not known. (FIG. 5B) In vitro translation in nondialyzed HeLa S10 extract without the addition of exogenous amino acids and tRNA and in the presence of competing cellular mRNAs uncovers a defect in translation capacities of codon-deoptimized PV genomes. Shown is a Western blot of poliovirus 2C reactive translation products (2C^ATPase, 2BC, and P2) resolved on a 10% SDS-PAGE gel. The relative amounts of the 2BC translation products are expressed below each lane as percentages of the wt band.

FIGS. 6A-B. Analysis of in vivo translation using dicistronic reporter replicons confirms the detrimental effect of codon deoptimization on PV translation. (FIG. 6A) Schematic of dicistronic replicons. Various P1 capsid coding sequences were inserted upstream of the firefly luciferase gene (F-Luc). Determination of changing levels of F-Luc expression relative to an internal control (R-Luc) allows for the quantification of ribosome transit through the P1 capsid region. (FIG. 6B) Replicon RNAs were transfected into HeLa cells and incubated for 7 h in the presence of 2 mM guanidine-hydrochloride to block RNA replication. The relative rate of translation through the P1 region was inversely proportional to the extent of codon deoptimization. While the capsid coding sequences of two viable virus constructs, PV-AB^2470-2954 and PV-AB^2954-3386, allow between 60 and 80% of wt translation, translation efficiency below 20% is associated with the lethal phenotypes observed with the PV-AB, PV-AB^2470-3386, and PV-AB^1513-2470 genomes. Values represents the average of 6 assays from 3 independent experiments.

FIG. 7. Determining codon pair bias of human and viral ORFs. Dots represent the average codon-pair score per codon pair for one ORF plotted against its length. Codon pair bias (CPB) was calculated for 14,795 annotated human genes. Under-represented codon pairs yield negative scores. CPB is plotted for various poliovirus P1 constructs, represented by symbols with arrows. The figure illustrates that the bulk of human genes clusters around 0.1. CPB is shown for PV(M)-wt (labeled “WT”) (−0.02), customized synthetic poliovirus capsids PV-Max (+0.25), PV-Min (−0.48), and PV(M)-wt:PV-Min chimera capsids PV-Min^755-2479 (=“PV-MinXY”) (−0.31) and PV-Min^2470-3386 (=“PV-MinZ”) (−0.20). Viruses PV-SD and PV-AB are the result of altered codon bias, but not altered codon pair bias.

FIGS. 8A-B. Characteristics of codon-pair deoptimized polio. (FIG. 8A) One-step growth kinetics reveals PFU production for PV-Min^755-2470 and PV-Min^2470-3385 that is reduced on the order of 2.5 orders of magnitude by comparison to PV(M)-wt. However, all viruses produce a similar number of viral particles (not shown in this Figure). (FIG. 8B) As a result the PFU/particle ratio is reduced, similar to codon deoptimized viruses PV-AB^755-1513 and PV-AB^2470-2954 (see FIG. 3B) (PFU is “Plaque Forming Unit”).

FIG. 9. Assembly of chimeric viral genomes. To “scan” through a target genome (red) small segments are amplified or synthesized and introduced into the wt genome (black) by overlapping PCR.

FIG. 10. The eight-plasmid pol I-pol II system for the generation of influenza A virus. Eight expression plasmids containing the eight viral cDNAs inserted between the human pol I promoter and the pol II promoter are transfected into eukaryotic cells. Because each plasmid contains two different promoters, both cellular pol I and pol II will transcribe the plasmid template, presumably in different nuclear compartments, which will result in the synthesis of viral mRNAs and vRNAs. After synthesis of the viral polymerase complex proteins (PB1, PB2, PA, nucleoproteins), the viral replication cycle is initiated. Ultimately, the assembly of all viral molecules directly (pol II transcription) or indirectly (pol I transcription and viral replication) derived from the cellular transcription and translation machinery results in the interaction of all synthesized molecules (vRNPs and the structural proteins HA, NA, M1, M2, NS2/NEP) to generate infectious influenza A virus. (Reproduced from Neumann et al., 2000.) (Note: there are other ways of synthesizing influenza de novo).

FIGS. 11A-B. Poliovirus Genome and Synthetic Viral Constructs. The poliovirus genome and open reading frames of chimeric virus constructs. (FIG. 11A) Top, a schematic of the full-length PV(M)-wt genomic RNA. (FIG. 11B) Below, the open reading frames of PV(M)-wt, the CPB customized synthetic viruses PV-Max, PV-Min, and the PV(M)-wt:PV-Min chimera viruses. Black corresponds to PV(M)-wt sequence, Gray to PV-Min synthetic sequence, and Thatched to PV-Max. The viral constructs highlighted, PV-Min^755-2470 (PV-MinXY) and PV-Min^2470-3385 (PV-MinZ), were further characterized due to a markedly attenuated phenotype.

FIGS. 12A-B. On-Step growth curves display similar kinetics yielding a similar quantity of particles with decreased infectivity. (FIG. 12A) An MOI of 2 was used to infect a monolayer of HeLa R19 cells, the PFU at the given time points (0, 2, 4, 7, 10, 24, 48 hrs) was measured by plaque assay. Corresponding symbols: (□) PV(M)-wt, (●) PV-Max, (⋄) PV-Min755-1513, (x) PV-Min1513-2470, (♦) PV-MinXY, (Δ) PV-MinZ (FIG. 12B) Displays the conversion of the calculated PFU/ml at each time point to particles/ml. This achieved by multiplying the PFU/ml by the respective viruses specific infectivity. Corresponding symbols as in (FIG. 12A)

FIGS. 13A-B. In vivo modulation of translation by alteration of CPB. (FIG. 13A) The dicistronic RNA construct used to quantify the in vivo effect CPB has on translation. The first cistron utilizes a hepatitis C virus (HCV) Internal Ribosome Entry Site (IRES) inducing the translation of Renilla Luciferase (R-Luc). This first cistron is the internal control used to normalize the amount of input RNA. The second cistron controlled by the PV(M)-wt IRES induces the translation of Firefly Luciferase (F-Luc). The region labeled “P1” in the construct was replaced by the cDNA of each respective viruses P1. (FIG. 13B) Each respective RNA construct was transfected, in the presence of 2 mM guanidine hydrochloride, into HeLa R19 cells and after 6 hours the R-Luc and F-Luc were measured. The F-Luc/R-Luc values were normalized relative to PV(M)-wt translation (100%).

FIG. 14. The heat inactivation profile of the synthetic viruses is unchanged. To rule out that large scale codon-pair bias modification alters the gross morphology of virions, as one might expect if capsid proteins were misfolded, the thermal stability of PVMinXY and PV-MinZ was tested. An equal number of particles were incubated at 50° C. and the remaining infectivity quantified after given periods of time via plaque assay. If the capsids of the synthetic viruses were destabilized we would expect increased loss of viability at 50° C. in comparison to wt PV(M). This was not the case. The thermal inactivation kinetics of both synthetic viruses was identical to the wt. In contrast, the Sabin-1 virus carries numerous mutations in the genome region encoding the capsid, which, fittingly, rendered this virus less heat stabile as compared to wt PV1(M).

FIG. 15. Neutralizing antibody titer following vaccination. A group of eight CD155 tg mice, seven of which completed the regimen, were each inoculated by intraperitoneal injection three times at weekly intervals with 10⁸ particles of PV-MinZ (●) and PV-MinXY (♦) and the serum conversion was measured 10 days after the final vaccination. A horizontal lines across each data set marks the average neutralizing antibody titer for each virus construct. The anti-poliovirus antibody titer was measured via micro-neutralization assay. (*) No virus neutralization for mock-vaccinated animals was detected at the lowest tested 1:8.

FIGS. 16A-B. Influenza virus carrying codon pair-deoptimized NP segment. (FIG. 16A) A/PR8-NP^Min virus are viable and produce smaller plaques on MDCK cells compared to the A/PR8 wt. (FIG. 16B) A/PR8-NP^Min virus display delayed growth kinetics and final titers 3-5 fold below wild type A/PR8.

FIGS. 17A-B. Influenza virus carrying codon pair-deoptimized PB1 or HA and NP segments. (FIG. 17A) A/PR8-PB1^Min-RR and A/PR8-HA^Min/NP^Min virus are viable and produce smaller plaques on MDCK cells as compared to the A/PR8 wild type. (FIG. 17B) A/PR8-PB1^Min-RR and A/PR8-HA^Min/NP^Min virus display delayed growth kinetics and final titers about 10 fold below wild type A/PR8.

FIGS. 18A-C. Attenuation of A/PR8-NP^Min in BALB/c mouse model. (FIG. 18A) A/PR8-NP^Min virus has reduced pathogenicity compared to wild type A/PR8 virus as determined by weight loss upon vaccination. (FIG. 18B) All mice (eight of eight) vaccinated with A/PR8-NP^Min virus survived, where as only 25% (two of eight) mice infected with A/PR8 were alive 13 days post vaccination. (FIG. 18C) Mice vaccinated with A/PR8-NP^Min virus are protected from challenge with 100×LD₅₀ of A/PR8 wild type virus.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the production of attenuated viruses that may be used as vaccines to protect against viral infection and disease. Accordingly, the invention provides an attenuated virus, which comprises a modified viral genome containing nucleotide substitutions engineered in multiple locations in the genome, wherein the substitutions introduce a plurality of synonymous codons into the genome and/or a change of the order of existing codons for the same amino acid (change of codon pair utilization). In both cases, the original, wild-type amino acid sequences of the viral gene products are retained.

Most amino acids are encoded by more than one codon. See the genetic code in Table 1. For instance, alanine is encoded by GCU, GCC, GCA, and GCG. Three amino acids (Leu, Ser, and Arg) are encoded by six different codons, while only Trp and Met have unique codons. “Synonymous” codons are codons that encode the same amino acid. Thus, for example, CUU, CUC, CUA, CUG, UUA, and UUG are synonymous codons that code for Leu. Synonymous codons are not used with equal frequency. In general, the most frequently used codons in a particular organism are those for which the cognate tRNA is abundant, and the use of these codons enhances the rate and/or accuracy of protein translation. Conversely, tRNAs for the rarely used codons are found at relatively low levels, and the use of rare codons is thought to reduce translation rate and/or accuracy. Thus, to replace a given codon in a nucleic acid by a synonymous but less frequently used codon is to substitute a “deoptimized” codon into the nucleic acid.

TABLE 1
Genetic Code
		U	C	A	G
	U	Phe	Ser	Tyr	Cys	U
		Phe	Ser	Tyr	Cys	C
		Leu	Ser	STOP	STOP	A
		Leu	Ser	STOP	Trp	G
	C	Leu	Pro	His	Arg	U
		Leu	Pro	His	Arg	C
		Leu	Pro	Gln	Arg	A
		Leu	Pro	Gln	Arg	G
	A	Ile	Thr	Asn	Ser	U
		Ile	Thr	Asn	Ser	C
		Ile	Thr	Lys	Arg	A
		Met	Thr	Lys	Arg	G
	G	Val	Ala	Asp	Gly	U
		Val	Ala	Asp	Gly	C
		Val	Ala	Glu	Gly	A
		Val	Ala	Glu	Gly	G
a The first nucleotide in each codon encoding a particular amino acid is shown in the left-most column; the second nucleotide is shown in the top row; and the third nucleotide is shown in the right-most column.

In addition, a given organism has a preference for the nearest codon neighbor of a given codon A, referred to a bias in codon pair utilization. A change of codon pair bias, without changing the existing codons, can influence the rate of protein synthesis and production of a protein.

In various embodiments of the present invention, the virus is a DNA, RNA, double-stranded, or single-stranded virus. In further embodiments, the virus infects an animal or a plant. In preferred embodiments, the animal is a human. A large number of animal viruses are well known to cause diseases (see below). Certain medically important viruses, such as those causing rabies, severe acute respiratory syndrome (SARS), and avian flu, can also spread to humans from their normal non-human hosts.

Viruses also constitute a major group of plant pathogens, and research is ongoing to develop viral vectors for producing transgenic plants. The advantages of such vectors include the ease of transforming plants, the ability to transform mature plants which obviates the need for regeneration of a transgenic plant from a single transformed cell, and high levels of expression of foreign genes from the multiple copies of virus per cell. However, one of the main disadvantages of these vectors is that it has not been possible to separate essential viral replicative functions from pathogenic determinants of the virus. The SAVE strategy disclosed herein may afford a means of engineering non-pathogenic viral vectors for plant transformation.

Major Viral Pathogens in Humans

Viral pathogens are the causative agents of many diseases in humans and other animals. Well known examples of viral diseases in humans include the common cold (caused by human rhinoviruses, HRV), influenza (influenza virus), chickenpox (varicella-zoster virus), measles (a paramyxovirus), mumps (a paramyxovirus), poliomyelitis (poliovirus, PV), rabies (Lyssavirus), cold sores (Herpes Simplex Virus [HSV] Type 1), and genital herpes (HSV Type 2). Prior to the introduction of vaccination programs for children, many of these were common childhood diseases worldwide, and are still a significant threat to health in some developing countries. Viral diseases also include more serious diseases such as acquired immunodeficiency syndrome (AIDS) caused by Human Immunodeficiency Virus (HIV), severe acute respiratory syndrome (SARS) caused by SARS coronavirus, avian flu (H5N1 subtype of influenza A virus), Ebola (ebolavirus), Marburg haemorrhagic fever (Marburg virus), dengue fever (Flavivirus serotypes), West Nile encephalitis (a flavivirus), infectious mononucleosis (Epstein-Barr virus, EBV), hepatitis (Hepatitis C Virus, HCV; hepatitis B virus, HBV), and yellow fever (flavivirus). Certain types of cancer can also be caused by viruses. For example, although most infections by human papillomavirus (HPV) are benign, HPV has been found to be associated with cervical cancer, and Kaposi's sarcoma (KS), a tumor prevalent in AIDS patients, is caused by Kaposi's sarcoma-associated herpesvirus (KSHV).

Because viruses reside within cells and use the machinery of the host cell to reproduce, they are difficult to eliminate without killing the host cell. The most effective approach to counter viral diseases has been the vaccination of subjects at risk of infection in order to provide resistance to infection. For some diseases (e.g., chickenpox, measles, mumps, yellow fever), effective vaccines are available. However, there is a pressing need to develop vaccines for many other viral diseases. The SAVE (Synthetic Attenuated Virus Engineering) approach to making vaccines described herein is in principle applicable to all viruses for which a reverse genetics system (see below) is available. This approach is exemplified herein by focusing on the application of SAVE to develop attenuated virus vaccines for poliomyelitis, the common cold, and influenza.

Any virus can be attenuated by the methods disclosed herein. The virus can be a dsDNA virus (e.g. Adenoviruses, Herpesviruses, Poxviruses), a single stranded “plus” sense DNA virus (e.g., Parvoviruses) a double stranded RNA virus (e.g., Reoviruses), a single stranded+sense RNA virus (e.g. Picornaviruses, Togaviruses), a single stranded “minus” sense RNA virus (e.g. Orthomyxoviruses, Rhabdoviruses), a single stranded+sense RNA virus with a DNA intermediate (e.g. Retroviruses), or a double stranded reverse transcribing virus (e.g. Hepadnaviruses). In certain non-limiting embodiments of the present invention, the virus is poliovirus (PV), rhinovirus, influenza virus including avian flu (e.g. H5N1 subtype of influenza A virus), severe acute respiratory syndrome (SARS) coronavirus, Human Immunodeficiency Virus (HIV), Hepatitis B Virus (HBV), Hepatitis C Virus (HCV), infectious bronchitis virus, ebolavirus, Marburg virus, dengue fever virus (Flavivirus serotypes), West Nile disease virus, Epstein-Barr virus (EBV), yellow fever virus, Ebola (ebolavirus), chickenpox (varicella-zoster virus), measles (a paramyxovirus), mumps (a paramyxovirus), rabies (Lyssavirus), human papillomavirus, Kaposi's sarcoma-associated herpesvirus, Herpes Simplex Virus (HSV Type 1), or genital herpes (HSV Type 2).

The term “parent” virus or “parent” protein encoding sequence is used herein to refer to viral genomes and protein encoding sequences from which new sequences, which may be more or less attenuated, are derived. Parent viruses and sequences are usually “wild type” or “naturally occurring” prototypes or isolates of variants for which it is desired to obtain a more highly attenuated virus. However, parent viruses also include mutants specifically created or selected in the laboratory on the basis of real or perceived desirable properties. Accordingly, parent viruses that are candidates for attenuation include mutants of wild type or naturally occurring viruses that have deletions, insertions, amion acid substitutions and the like, and also include mutants which have codon substitutions. In one embodiment, such a parent sequence differs from a natural isolate by about 30 amino acids or fewer. In another embodiment, the parent sequence differs from a natural isolate by about 20 amino acids or fewer. In yet another embodiment, the parent sequence differs from a natural isolate by about 10 amino acids or fewer.

The attenuated PV may be derived from poliovirus type 1 (Mahoney; “PV(M)”), poliovirus type 2 (Lansing), poliovirus type 3 (Leon), monovalent oral poliovirus vaccine (OPV) virus, or trivalent OPV virus. In certain embodiments, the poliovirus is PV-AB having the genomic sequence set forth in SEQ ID NO:2, or PV-AB^755-1513, PV-AB^755-2470, PV-AB^1513-3386, PV-AB^2470-3386, PV-AB^1513-2470, PV-AB^2470-2954, or PV-AB^2954-3386. The nomenclature reflects a PV(M) genome in which portions of the genome, are substituted with nucleotides of PV-AB. The superscript provides the nucleotide numbers of PV-AB that are substituted.

In various embodiments, the attenuated rhinovirus is a human rhinovirus (HRV) derived from HRV2, HRV14, Human rhinovirus 10 Human rhinovirus 100; Human rhinovirus 11; Human rhinovirus 12; Human rhinovirus 13; Human rhinovirus 15; Human rhinovirus 16; Human rhinovirus 18; Human rhinovirus 19; Human rhinovirus 1A; Human rhinovirus 1B; Human rhinovirus 2; Human rhinovirus 20; Human rhinovirus 21; Human rhinovirus 22; Human rhinovirus 23; Human rhinovirus 24; Human rhinovirus 25; Human rhinovirus 28; Human rhinovirus 29; Human rhinovirus 30; Human rhinovirus 31 Human rhinovirus 32; Human rhinovirus 33; Human rhinovirus 34; Human rhinovirus 36; Human rhinovirus 38; Human rhinovirus 39; Human rhinovirus 40; Human rhinovirus 41; Human rhinovirus 43; Human rhinovirus 44; Human rhinovirus 45; Human rhinovirus 46; Human rhinovirus 47; Human rhinovirus 49; Human rhinovirus 50; Human rhinovirus 51; Human rhinovirus 53; Human rhinovirus 54; Human rhinovirus 55; Human rhinovirus 56; Human rhinovirus 57; Human rhinovirus 58; Human rhinovirus 59; Human rhinovirus 60; Human rhinovirus 61; Human rhinovirus 62; Human rhinovirus 63; Human rhinovirus 64; Human rhinovirus 65; Human rhinovirus 66; Human rhinovirus 67; Human rhinovirus 68; Human rhinovirus 7; Human rhinovirus 71; Human rhinovirus 73; Human rhinovirus 74; Human rhinovirus 75; Human rhinovirus 76; Human rhinovirus 77; Human rhinovirus 78; Human rhinovirus 8; Human rhinovirus 80; Human rhinovirus 81; Human rhinovirus 82; Human rhinovirus 85; Human rhinovirus 88; Human rhinovirus 89; Human rhinovirus 9; Human rhinovirus 90; Human rhinovirus 94; Human rhinovirus 95; Human rhinovirus 96 Human rhinovirus 98; Human rhinovirus 14; Human rhinovirus 17; Human rhinovirus 26; Human rhinovirus 27; Human rhinovirus 3; Human rhinovirus 8001 Finland November 1995; Human rhinovirus 35; Human rhinovirus 37; +Human rhinovirus 6253 Finland September 1994; Human rhinovirus 9166 Finland September 1995; Human rhinovirus 4; Human rhinovirus 42; Human rhinovirus 48; Human rhinovirus 9864 Finland September 1996; Human rhinovirus 5; Human rhinovirus 52; Human rhinovirus 6; Human rhinovirus 7425 Finland December 1995; Human rhinovirus 69; Human rhinovirus 5928 Finland May 1995; Human rhinovirus 70; Human rhinovirus 72; Human rhinovirus 79; Human rhinovirus 83; Human rhinovirus 84; Human rhinovirus 8317 Finland August 1996; Human rhinovirus 86; Human rhinovirus 91; Human rhinovirus 7851 Finland September 1996; Human rhinovirus 92; Human rhinovirus 93; Human rhinovirus 97; Human rhinovirus 99; Antwerp rhinovirus 98/99; Human rhinovirus 263 Berlin 2004; Human rhinovirus 3083/rhino/Hyogo/2005; Human rhinovirus NY-003; Human rhinovirus NY-028; Human rhinovirus NY-041; Human rhinovirus NY-042; Human rhinovirus NY-060; Human rhinovirus NY-063; Human rhinovirus NY-074; Human rhinovirus NY-1085; Human rhinovirus strain Hanks; Untyped human rhinovirus OK88-8162; Human enterovirus sp. ex Amblyomma americanum; Human rhinovirus sp. or Human rhinovirus UC.

In other embodiments, the attenuated influenza virus is derived from influenza virus A, influenza virus B, or influenza virus C. In further embodiments, the influenza virus A belongs to but is not limited to subtype H10N7, H10N1, H10N2, H10N3, H10N4, H10N5, H10N6, H10N7, H10N8, H10N9, H11N1, H11N2, H11N3, H11N4, H11N6, H11N8, H11N9, H12N1, H12N2, H12N4, H12N5, H12N6, H12N8, H12N9, H13N2, H13N3, H13N6, H13N9, H14N5, H14N6, H15N2, H15N8, H15N9, H16N3, H1N1, H1N2, H1N3, H1N5, H1N6, H1N8, H1N9, H2N1, H2N2, H2N3, H2N4, H2N5, H2N6, H2N7, H2N8, H2N9, H3N1, H3N2, H3N3, H3N4, H3N5, H3N6, H3N8, H3N9, H4N1, H4N2, H4N3, H4N4, H4N5, H4N6, H4N7, H4N8, H4N9, H5N1, H5N2, H5N3, H5N4, H5N6, H5N7, H5N8, H5N9, H6N1, H6N2, H6N3, H6N4, H6N5, H6N6, H6N7, H6N8, H6N9, H7N1, H7N2, H7N3, H7N4, H7N5, H7N7, H7N8, H7N9, H8N2, H8N4, H8N5, H9N1, H9N2, H9N3, H9N4, H9N5, H9N6, H9N7, H9N8, H9N9 and unidentified subtypes.

In further embodiments, the influenza virus B belongs to but is not limited to subtype Influenza B virus (B/Aichi/186/2005), Influenza B virus (B/Aichi/5/88), Influenza B virus (B/Akita/27/2001), Influenza B virus (B/Akita/5/2001), Influenza B virus (B/Alabama/1/2006), Influenza B virus (B/Alabama/2/2005), Influenza B virus (B/Alaska/03/1992), Influenza B virus (B/Alaska/12/1996), Influenza B virus (B/Alaska/16/2000), Influenza B virus (B/Alaska/16/2003), Influenza B virus (B/Alaska/1777/2005), Influenza B virus (B/Alaska/2/2004), Influenza B virus (B/Alaska/6/2005), Influenza B virus (B/Ann Arbor/1/1986), Influenza B virus (B/Ann Arbor/1994), Influenza B virus (B/Argentina/132/2001), Influenza B virus (B/Argentina/3640/1999), Influenza B virus (B/Argentina/69/2001), Influenza B virus (B/Arizona/1/2005), Influenza B virus (B/Arizona/12/2003), Influenza B virus (B/Arizona/13/2003), Influenza B virus (B/Arizona/135/2005), Influenza B virus (B/Arizona/14/2001), Influenza B virus (B/Arizona/14/2005), Influenza B virus (B/Arizona/140/2005), Influenza B virus (B/Arizona/146/2005), Influenza B virus (B/Arizona/148/2005), Influenza B virus (B/Arizona/15/2005), Influenza B virus (B/Arizona/16/2005), Influenza B virus (B/Arizona/162/2005), Influenza B virus (B/Arizona/163/2005), Influenza B virus (B/Arizona/164/2005), Influenza B virus (B/Arizona/2/2000), Influenza B virus (B/Arizona/2/2005), Influenza B virus (B/Arizona/2e/2006), Influenza B virus (B/Arizona/3/2006), Influenza B virus (B/Arizona/4/2002), Influenza B virus (B/Arizona/4/2006), Influenza B virus (B/Arizona/48/2005), Influenza B virus (B/Arizona/5/2000), Influenza B virus (B/Arizona/59/2005), Influenza B virus (B/Arizona/7/2000), Influenza B virus (B/Auckland/01/2000), Influenza B virus (B/Bangkok/141/1994), Influenza B virus (B/Bangkok/143/1994), Influenza B virus (B/Bangkok/153/1990), Influenza B virus (B/Bangkok/163/1990), Influenza B virus (B/Bangkok/163/90), Influenza B virus (B/Bangkok/34/99), Influenza B virus (B/Bangkok/460/03), Influenza B virus (B/Bangkok/54/99), Influenza B virus (B/Barcelona/215/03), Influenza B virus (B/Beijing/15/84), Influenza B virus (B/Beijing/184/93), Influenza B virus (B/Beijing/243/97), Influenza B virus (B/Beijing/43/75), Influenza B virus (B/Beijing/5/76), Influenza B virus (B/Beijing/76/98), Influenza B virus (B/Belgium/WV106/2002), Influenza B virus (B/Belgium/WV107/2002), Influenza B virus (B/Belgium/WV109/2002), Influenza B virus (B/Belgium/WV114/2002), Influenza B virus (B/Belgium/WV122/2002), Influenza B virus (B/Bonn/43), Influenza B virus (B/Brazil/017/00), Influenza B virus (B/Brazil/053/00), Influenza B virus (B/Brazil/055/00), Influenza B virus (B/Brazil/064/00), Influenza B virus (B/Brazil/074/00), Influenza B virus (B/Brazil/079/00), Influenza B virus (B/Brazil/110/01), Influenza B virus (B/Brazil/952/2001), Influenza B virus (B/Brazil/975/2000), Influenza B virus (B/Brisbane/32/2002), Influenza B virus (B/Bucharest/311/1998), Influenza B virus (B/Bucharest/795/03), Influenza B virus (B/Buenos Aires/161/00), Influenza B virus (B/Buenos Aires/9/95), Influenza B virus (B/Buenos Aires/SW16/97), Influenza B virus (B/Buenos Aires/VL518/99), Influenza B virus (B/California/01/1995), Influenza B virus (B/California/02/1994), Influenza B virus (B/California/02/1995), Influenza B virus (B/California/1/2000), Influenza B virus (B/California/10/2000), Influenza B virus (B/California/11/2001), Influenza B virus (B/California/14/2005), Influenza B virus (B/California/2/2002), Influenza B virus (B/California/2/2003), Influenza B virus (B/California/3/2000), Influenza B virus (B/California/3/2004), Influenza B virus (B/California/6/2000), Influenza B virus (B/California/7/2005), Influenza B virus (B/Canada/16188/2000), Influenza B virus (B/Canada/464/2001), Influenza B virus (B/Canada/464/2002), Influenza B virus (B/Chaco/366/00), Influenza B virus (B/Chaco/R113/00), Influenza B virus (B/Chantaburi/218/2003), Influenza B virus (B/Cheju/303/03), Influenza B virus (B/Chiba/447/98), Influenza B virus (B/Chile/3162/2002), Influenza B virus (B/Chongqing/3/2000), Influenza B virus (B/clinical isolate SA1 Thailand/2002), Influenza B virus (B/clinical isolate SA10 Thailand/2002), Influenza B virus (B/clinical isolate SA100 Philippines/2002), Influenza B virus (B/clinical isolate SA101 Philippines/2002), Influenza B virus (B/clinical isolate SA102 Philippines/2002), Influenza B virus (B/clinical isolate SA103 Philippines/2002), Influenza B virus (B/clinical isolate SA104 Philippines/2002), Influenza B virus (B/clinical isolate SA105 Philippines/2002), Influenza B virus (B/clinical isolate SA106 Philippines/2002), Influenza B virus (B/clinical isolate SA107 Philippines/2002), Influenza B virus (B/clinical isolate SA108 Philippines/2002), Influenza B virus (B/clinical isolate SA109 Philippines/2002), Influenza B virus (B/clinical isolate SA11 Thailand/2002), Influenza B virus (B/clinical isolate SA110 Philippines/2002), Influenza B virus (B/clinical isolate SA112 Philippines/2002), Influenza B virus (B/clinical isolate SA113 Philippines/2002), Influenza B virus (B/clinical isolate SA114 Philippines/2002), Influenza B virus (B/clinical isolate SA115 Philippines/2002), Influenza B virus (B/clinical isolate SA116 Philippines/2002), Influenza B virus (B/clinical isolate SA12 Thailand/2002), Influenza B virus (B/clinical isolate SA13 Thailand/2002), Influenza B virus (B/clinical isolate SA14 Thailand/2002), Influenza B virus (B/clinical isolate SA15 Thailand/2002), Influenza B virus (B/clinical isolate SA16 Thailand/2002), Influenza B virus (B/clinical isolate SA17 Thailand/2002), Influenza B virus (B/clinical isolate SA18 Thailand/2002), Influenza B virus (B/clinical isolate SA19 Thailand/2002), Influenza B virus (B/clinical isolate SA2 Thailand/2002), Influenza B virus (B/clinical isolate SA20 Thailand/2002), Influenza B virus (B/clinical isolate SA21 Thailand/2002), Influenza B virus (B/clinical isolate SA22 Thailand/2002), Influenza B virus (B/clinical isolate SA23 Thailand/2002), Influenza B virus (B/clinical isolate SA24 Thailand/2002), Influenza B virus (B/clinical isolate SA25 Thailand/2002), Influenza B virus (B/clinical isolate SA26 Thailand/2002), Influenza B virus (B/clinical isolate SA27 Thailand/2002), Influenza B virus (B/clinical isolate SA28 Thailand/2002), Influenza B virus (B/clinical isolate SA29 Thailand/2002), Influenza B virus (B/clinical isolate SA3 Thailand/2002), Influenza B virus (B/clinical isolate SA30 Thailand/2002), Influenza B virus (B/clinical isolate SA31 Thailand/2002), Influenza B virus (B/clinical isolate SA32 Thailand/2002), Influenza B virus (B/clinical isolate SA33 Thailand/2002), Influenza B virus (B/clinical isolate SA34 Thailand/2002), Influenza B virus (B/clinical isolate SA37 Thailand/2002), Influenza B virus (B/clinical isolate SA38 Philippines/2002), Influenza B virus (B/clinical isolate SA39 Thailand/2002), Influenza B virus (B/clinical isolate SA40 Thailand/2002), Influenza B virus (B/clinical isolate SA41 Philippines/2002), Influenza B virus (B/clinical isolate SA42 Philippines/2002), Influenza B virus (B/clinical isolate SA43 Thailand/2002), Influenza B virus (B/clinical isolate SA44 Thailand/2002), Influenza B virus (B/clinical isolate SA45 Philippines/2002), Influenza B virus (B/clinical isolate SA46 Philippines/2002), Influenza B virus (B/clinical isolate SA47 Philippines/2002), Influenza B virus (B/clinical isolate SA5 Thailand/2002), Influenza B virus (B/clinical isolate SA50 Philippines/2002), Influenza B virus (B/clinical isolate SA51 Philippines/2002), Influenza B virus (B/clinical isolate SA52 Philippines/2002), Influenza B virus (B/clinical isolate SA53 Philippines/2002), Influenza B virus (B/clinical isolate SA57 Philippines/2002), Influenza B virus (B/clinical isolate SA58 Philippines/2002), Influenza B virus (B/clinical isolate SA59 Philippines/2002), Influenza B virus (B/clinical isolate SA6 Thailand/2002), Influenza B virus (B/clinical isolate SA60 Philippines/2002), Influenza B virus (B/clinical isolate SA61 Philippines/2002), Influenza B virus (B/clinical isolate SA62 Philippines/2002), Influenza B virus (B/clinical isolate SA63 Philippines/2002), Influenza B virus (B/clinical isolate SA64 Philippines/2002), Influenza B virus (B/clinical isolate SA65 Philippines/2002), Influenza B virus (B/clinical isolate SA66 Philippines/2002), Influenza B virus (B/clinical isolate SA67 Philippines/2002), Influenza B virus (B/clinical isolate SA68 Philippines/2002), Influenza B virus (B/clinical isolate SA69 Philippines/2002), Influenza B virus (B/clinical isolate SA7 Thailand/2002), Influenza B virus (B/clinical isolate SA70 Philippines/2002), Influenza B virus (B/clinical isolate SA71 Philippines/2002), Influenza B virus (B/clinical isolate SA73 Philippines/2002), Influenza B virus (B/clinical isolate SA74 Philippines/2002), Influenza B virus (B/clinical isolate SA76 Philippines/2002), Influenza B virus (B/clinical isolate SA77 Philippines/2002), Influenza B virus (B/clinical isolate SA78 Philippines/2002), Influenza B virus (B/clinical isolate SA79 Philippines/2002), Influenza B virus (B/clinical isolate SA8 Thailand/2002), Influenza B virus (B/clinical isolate SA80 Philippines/2002), Influenza B virus (B/clinical isolate SA81 Philippines/2002), Influenza B virus (B/clinical isolate SA82 Philippines/2002), Influenza B virus (B/clinical isolate SA83 Philippines/2002), Influenza B virus (B/clinical isolate SA84 Philippines/2002), Influenza B virus (B/clinical isolate SA85 Thailand/2002), Influenza B virus (B/clinical isolate SA86 Thailand/2002), Influenza B virus (B/clinical isolate SA87 Thailand/2002), Influenza B virus (B/clinical isolate SA88 Thailand/2002), Influenza B virus (B/clinical isolate SA89 Thailand/2002), Influenza B virus (B/clinical isolate SA9 Thailand/2002), Influenza B virus (B/clinical isolate SA90 Thailand/2002), Influenza B virus (B/clinical isolate SA91 Thailand/2002), Influenza B virus (B/clinical isolate SA92 Thailand/2002), Influenza B virus (B/clinical isolate SA93 Thailand/2002), Influenza B virus (B/clinical isolate SA94 Thailand/2002), Influenza B virus (B/clinical isolate SA95 Philippines/2002), Influenza B virus (B/clinical isolate SA96 Thailand/2002), Influenza B virus (B/clinical isolate SA97 Philippines/2002), Influenza B virus (B/clinical isolate SA98 Philippines/2002), Influenza B virus (B/clinical isolate SA99 Philippines/2002), Influenza B virus (B/CNIC/27/2001), Influenza B virus (B/Colorado/04/2004), Influenza B virus (B/Colorado/11e/2004), Influenza B virus (B/Colorado/12e/2005), Influenza B virus (B/Colorado/13/2004), Influenza B virus (B/Colorado/13e/2004), Influenza B virus (B/Colorado/15/2004), Influenza B virus (B/Colorado/16e/2004), Influenza B virus (B/Colorado/17e/2004), Influenza B virus (B/Colorado/2/2004), Influenza B virus (B/Colorado/2597/2004), Influenza B virus (B/Colorado/4e/2004), Influenza B virus (B/Colorado/5/2004), Influenza B virus (B/Connecticut/02/1995), Influenza B virus (B/Connecticut/07/1993), Influenza B virus (B/Cordoba/2979/1991), Influenza B virus (B/Cordoba/VA418/99), Influenza B virus (B/Czechoslovakia/16/89), Influenza B virus (B/Czechoslovakia/69/1990), Influenza B virus (B/Czechoslovakia/69/90), Influenza B virus (B/Daeku/10/97), Influenza B virus (B/Daeku/45/97), Influenza B virus (B/Daeku/47/97), Influenza B virus (B/Daeku/9/97), Influenza B virus (B/Delaware/1/2006), Influenza B virus (B/Du/4/78), Influenza B virus (B/Durban/39/98), Influenza B virus (B/Durban/43/98), Influenza B virus (B/Durban/44/98), Influenza B virus (B/Durban/52/98), Influenza B virus (B/Durban/55/98), Influenza B virus (B/Durban/56/98), Influenza B virus (B/Egypt/2040/2004), Influenza B virus (B/England/1716/2005), Influenza B virus (B/England/2054/2005), Influenza B virus (B/England/23/04), Influenza B virus (B/EspiritoSanto/55/01), Influenza B virus (B/EspiritoSanto/79/99), Influenza B virus (B/Finland/154/2002), Influenza B virus (B/Finland/159/2002), Influenza B virus (B/Finland/160/2002), Influenza B virus (B/Finland/161/2002), Influenza B virus (B/Finland/162/03), Influenza B virus (B/Finland/162/2002), Influenza B virus (B/Finland/162/91), Influenza B virus (B/Finland/164/2003), Influenza B virus (B/Finland/172/91), Influenza B virus (B/Finland/173/2003), Influenza B virus (B/Finland/176/2003), Influenza B virus (B/Finland/184/91), Influenza B virus (B/Finland/188/2003), Influenza B virus (B/Finland/190/2003), Influenza B virus (B/Finland/191/2003), Influenza B virus (B/Finland/192/2003), Influenza B virus (B/Finland/193/2003), Influenza B virus (B/Finland/199/2003), Influenza B virus (B/Finland/202/2003), Influenza B virus (B/Finland/203/2003), Influenza B virus (B/Finland/204/2003), Influenza B virus (B/Finland/205/2003), Influenza B virus (B/Finland/206/2003), Influenza B virus (B/Finland/220/2003), Influenza B virus (B/Finland/223/2003), Influenza B virus (B/Finland/225/2003), Influenza B virus (B/Finland/227/2003), Influenza B virus (B/Finland/231/2003), Influenza B virus (B/Finland/235/2003), Influenza B virus (B/Finland/239/2003), Influenza B virus (B/Finland/244/2003), Influenza B virus (B/Finland/245/2003), Influenza B virus (B/Finland/254/2003), Influenza B virus (B/Finland/254/93), Influenza B virus (B/Finland/255/2003), Influenza B virus (B/Finland/260/93), Influenza B virus (B/Finland/268/93), Influenza B virus (B/Finland/270/2003), Influenza B virus (B/Finland/275/2003), Influenza B virus (B/Finland/767/2000), Influenza B virus (B/Finland/84/2002), Influenza B virus (B/Finland/886/2001), Influenza B virus (B/Finland/WV4/2002), Influenza B virus (B/Finland/WV5/2002), Influenza B virus (B/Florida/02/1998), Influenza B virus (B/Florida/02/2006), Influenza B virus (B/Florida/1/2000), Influenza B virus (B/Florida/1/2004), Influenza B virus (B/Florida/2/2004), Influenza B virus (B/Florida/2/2005), Influenza B virus (B/Florida/2/2006), Influenza B virus (B/Florida/7e/2004), Influenza B virus (B/Fujian/36/82), Influenza B virus (B/Geneva/5079/03), Influenza B virus (B/Genoa/11/02), Influenza B virus (B/Genoa/2/02), Influenza B virus (B/Genoa/21/02), Influenza B virus (B/Genoa/33/02), Influenza B virus (B/Genoa/41/02), Influenza B virus (B/Genoa/52/02), Influenza B virus (B/Genoa/55/02), Influenza B virus (B/Genoa/56/02), Influenza B virus (B/Genoa/7/02), Influenza B virus (B/Genoa/8/02), Influenza B virus (B/Genoa12/02), Influenza B virus (B/Genoa3/02), Influenza B virus (B/Genoa48/02), Influenza B virus (B/Genoa49/02), Influenza B virus (B/Genoa5/02), Influenza B virus (B/Genoa53/02), Influenza B virus (B/Genoa6/02), Influenza B virus (B/Genoa65/02), Influenza B virus (B/Genova/1294/03), Influenza B virus (B/Genova/1603/03), Influenza B virus (B/Genova/2/02), Influenza B virus (B/Genova/20/02), Influenza B virus (B/Genova/2059/03), Influenza B virus (B/Genova/26/02), Influenza B virus (B/Genova/30/02), Influenza B virus (B/Genova/54/02), Influenza B virus (B/Genova/55/02), Influenza B virus (B/Georgia/02/1998), Influenza B virus (B/Georgia/04/1998), Influenza B virus (B/Georgia/09/2005), Influenza B virus (B/Georgia/1/2000), Influenza B virus (B/Georgia/1/2005), Influenza B virus (B/Georgia/2/2005), Influenza B virus (B/Georgia/9/2005), Influenza B virus (B/Guangdong/05/94), Influenza B virus (B/Guangdong/08/93), Influenza B virus (B/Guangdong/5/94), Influenza B virus (B/Guangdong/55/89), Influenza B virus (B/Guangdong/8/93), Influenza B virus (B/Guangzhou/7/97), Influenza B virus (B/Guangzhou/86/92), Influenza B virus (B/Guangzhou/87/92), Influenza B virus (B/Gyeonggi/592/2005), Influenza B virus (B/Hannover/2/90), Influenza B virus (B/Harbin/07/94), Influenza B virus (B/Hawaii/1/2003), Influenza B virus (B/Hawaii/10/2001), Influenza B virus (B/Hawaii/10/2004), Influenza B virus (B/Hawaii/11/2004), Influenza B virus (B/Hawaii/11e/2004), Influenza B virus (B/Hawaii/11e/2005), Influenza B virus (B/Hawaii/12e/2005), Influenza B virus (B/Hawaii/13/2004), Influenza B virus (B/Hawaii/13e/2004), Influenza B virus (B/Hawaii/17/2001), Influenza B virus (B/Hawaii/18e/2004), Influenza B virus (B/Hawaii/1990/2004), Influenza B virus (B/Hawaii/1993/2004), Influenza B virus (B/Hawaii/19e/2004), Influenza B virus (B/Hawaii/2/2000), Influenza B virus (B/Hawaii/2/2003), Influenza B virus (B/Hawaii/20e/2004), Influenza B virus (B/Hawaii/21/2004), Influenza B virus (B/Hawaii/26/2001), Influenza B virus (B/Hawaii/31e/2004), Influenza B virus (B/Hawaii/32e/2004), Influenza B virus (B/Hawaii/33e/2004), Influenza B virus (B/Hawaii/35/2001), Influenza B virus (B/Hawaii/36/2001), Influenza B virus (B/Hawaii/37/2001), Influenza B virus (B/Hawaii/38/2001), Influenza B virus (B/Hawaii/4/2006), Influenza B virus (B/Hawaii/43/2001), Influenza B virus (B/Hawaii/44/2001), Influenza B virus (B/Hawaii/9/2001), Influenza B virus (B/Hebei/19/94), Influenza B virus (B/Hebei/3/94), Influenza B virus (B/Hebei/4/95), Influenza B virus (B/Henan/22/97), Influenza B virus (B/Hiroshima/23/2001), Influenza B virus (B/Hong Kong/02/1993), Influenza B virus (B/Hong Kong/03/1992), Influenza B virus (B/Hong Kong/05/1972), Influenza B virus (B/Hong Kong/06/2001), Influenza B virus (B/Hong Kong/110/99), Influenza B virus (B/Hong Kong/1115/2002), Influenza B virus (B/Hong Kong/112/2001), Influenza B virus (B/Hong Kong/123/2001), Influenza B virus (B/Hong Kong/1351/02), Influenza B virus (B/Hong Kong/1351/2002), Influenza B virus (B/Hong Kong/1434/2002), Influenza B virus (B/Hong Kong/147/99), Influenza B virus (B/Hong Kong/156/99), Influenza B virus (B/Hong Kong/157/99), Influenza B virus (B/Hong Kong/167/2002), Influenza B virus (B/Hong Kong/22/1989), Influenza B virus (B/Hong Kong/22/2001), Influenza B virus (B/Hong Kong/22/89), Influenza B virus (B/Hong Kong/28/2001), Influenza B virus (B/Hong Kong/293/02), Influenza B virus (B/Hong Kong/310/2004), Influenza B virus (B/Hong Kong/329/2001), Influenza B virus (B/Hong Kong/330/2001 egg adapted), Influenza B virus (B/Hong Kong/330/2001), Influenza B virus (B/Hong Kong/330/2002), Influenza B virus (B/Hong Kong/335/2001), Influenza B virus (B/Hong Kong/336/2001), Influenza B virus (B/Hong Kong/497/2001), Influenza B virus (B/Hong Kong/542/2000), Influenza B virus (B/Hong Kong/548/2000), Influenza B virus (B/Hong Kong/553a/2003), Influenza B virus (B/Hong Kong/557/2000), Influenza B virus (B/Hong Kong/6/2001), Influenza B virus (B/Hong Kong/666/2001), Influenza B virus (B/Hong Kong/692/01), Influenza B virus (B/Hong Kong/70/1996), Influenza B virus (B/Hong Kong/8/1973), Influenza B virus (B/Hong Kong/9/89), Influenza B virus (B/Houston/1/91), Influenza B virus (B/Houston/1/92), Influenza B virus (B/Houston/1/96), Influenza B virus (B/Houston/2/93), Influenza B virus (B/Houston/2/96), Influenza B virus (B/Houston/B15/1999), Influenza B virus (B/Houston/B56/1997), Influenza B virus (B/Houston/B57/1997), Influenza B virus (B/Houston/B58/1997), Influenza B virus (B/Houston/B59/1997), Influenza B virus (B/Houston/B60/1997), Influenza B virus (B/Houston/B61/1997), Influenza B virus (B/Houston/B63/1997), Influenza B virus (B/Houston/B65/1998), Influenza B virus (B/Houston/B66/2000), Influenza B virus (B/Houston/B67/2000), Influenza B virus (B/Houston/B68/2000), Influenza B virus (B/Houston/B69/2002), Influenza B virus (B/Houston/B70/2002), Influenza B virus (B/Houston/B71/2002), Influenza B virus (B/Houston/B720/2004), Influenza B virus (B/Houston/B74/2002), Influenza B virus (B/Houston/B745/2005), Influenza B virus (B/Houston/B75/2002), Influenza B virus (B/Houston/B756/2005), Influenza B virus (B/Houston/B77/2002), Influenza B virus (B/Houston/B787/2005), Influenza B virus (B/Houston/B79/2003), Influenza B virus (B/Houston/B81/2003), Influenza B virus (B/Houston/B84/2003), Influenza B virus (B/Houston/B846/2005), Influenza B virus (B/Houston/B850/2005), Influenza B virus (B/Houston/B86/2003), Influenza B virus (B/Houston/B87/2003), Influenza B virus (B/Houston/B88/2003), Influenza B virus (B/Hunan/4/72), Influenza B virus (B/Ibaraki/2/85), Influenza B virus (B/Idaho/1/2005), Influenza B virus (B/Illinois/1/2004), Influenza B virus (B/Illinois/13/2004), Influenza B virus (B/Illinois/13/2005), Influenza B virus (B/Illinois/13e/2005), Influenza B virus (B/Illinois/3/2001), Influenza B virus (B/Illinois/3/2005), Influenza B virus (B/Illinois/33/2005), Influenza B virus (B/Illinois/36/2005), Influenza B virus (B/Illinois/4/2005), Influenza B virus (B/Illinois/47/2005), Influenza B virus (B/Incheon/297/2005), Influenza B virus (B/India/3/89), Influenza B virus (B/India/7526/2001), Influenza B virus (B/India/7569/2001), Influenza B virus (B/India/7600/2001), Influenza B virus (B/India/7605/2001), Influenza B virus (B/India/77276/2001), Influenza B virus (B/Indiana/01/1995), Influenza B virus (B/Indiana/3/2006), Influenza B virus (B/Indiana/5/2006), Influenza B virus (B/Iowa/03/2002), Influenza B virus (B/Iowa/1/2001), Influenza B virus (B/Iowa/1/2005), Influenza B virus (B/Israel/95/03), Influenza B virus (B/Israel/WV124/2002), Influenza B virus (B/Israel/WV126/2002), Influenza B virus (B/Israel/WV133/2002), Influenza B virus (B/Israel/WV135/2002), Influenza B virus (B/Israel/WV137/2002), Influenza B virus (B/Israel/WV142/2002), Influenza B virus (B/Israel/WV143/2002), Influenza B virus (B/Israel/WV145/2002), Influenza B virus (B/Israel/WV146/2002), Influenza B virus (B/Israel/WV150/2002), Influenza B virus (B/Israel/WV153/2002), Influenza B virus (B/Israel/WV158/2002), Influenza B virus (B/Israel/WV161/2002), Influenza B virus (B/Israel/WV166/2002), Influenza B virus (B/Israel/WV169/2002), Influenza B virus (B/Israel/WV170/2002), Influenza B virus (B/Israel/WV174/2002), Influenza B virus (B/Israel/WV183/2002), Influenza B virus (B/Israel/WV187/2002), Influenza B virus (B/Istanbul/CTF-132/05), Influenza B virus (B/Japan/1224/2005), Influenza B virus (B/Japan/1905/2005), Influenza B virus (B/Jiangsu/10/03), Influenza B virus (B/Jiangsu/10/2003 (recomb)), Influenza B virus (B/Jiangsu/10/2003), Influenza B virus (B/Jilin/20/2003), Influenza B virus (B/Johannesburg/05/1999), Influenza B virus (B/Johannesburg/06/1994), Influenza B virus (B/Johannesburg/1/99), Influenza B virus (B/Johannesburg/113/010), Influenza B virus (B/Johannesburg/116/01), Influenza B virus (B/Johannesburg/119/01), Influenza B virus (B/Johannesburg/123/01), Influenza B virus (B/Johannesburg/163/99), Influenza B virus (B/Johannesburg/187/99), Influenza B virus (B/Johannesburg/189/99), Influenza B virus (B/Johannesburg/2/99), Influenza B virus (B/Johannesburg/27/2005), Influenza B virus (B/Johannesburg/33/01), Influenza B virus (B/Johannesburg/34/01), Influenza B virus (B/Johannesburg/35/01), Influenza B virus (B/Johannesburg/36/01), Influenza B virus (B/Johannesburg/41/99), Influenza B virus (B/Johannesburg/5/99), Influenza B virus (B/Johannesburg/69/2001), Influenza B virus (B/Johannesburg/77/01), Influenza B virus (B/Johannesburg/94/99), Influenza B virus (B/Johannesburg/96/01), Influenza B virus (B/Kadoma/1076/99), Influenza B virus (B/Kadoma/122/99), Influenza B virus (B/Kadoma/122/99-V1), Influenza B virus (B/Kadoma/122/99-V10), Influenza B virus (B/Kadoma/122/99-V11), Influenza B virus (B/Kadoma/122/99-V2), Influenza B virus (B/Kadoma/122/99-V3), Influenza B virus (B/Kadoma/122/99-V4), Influenza B virus (B/Kadoma/122/99-V5), Influenza B virus (B/Kadoma/122/99-V6), Influenza B virus (B/Kadoma/122/99-V7), Influenza B virus (B/Kadoma/122/99-V8), Influenza B virus (B/Kadoma/122/99-V9), Influenza B virus (B/Kadoma/136/99), Influenza B virus (B/Kadoma/409/2000), Influenza B virus (B/Kadoma/506/99), Influenza B virus (B/kadoma/642/99), Influenza B virus (B/Kadoma/647/99), Influenza B virus (B/Kagoshima/15/94), Influenza B virus (B/Kanagawa/73), Influenza B virus (B/Kansas/1/2005), Influenza B virus (B/Kansas/22992/99), Influenza B virus (B/Kentucky/4/2005), Influenza B virus (B/Khazkov/224/91), Influenza B virus (B/Kisumu/2036/2006), Influenza B virus (B/Kisumu/2037/2006), Influenza B virus (B/Kisumu/2038/2006), Influenza B virus (B/Kisumu/2039/2006), Influenza B virus (B/Kisumu/2040/2006), Influenza B virus (B/Kisumu/7/2005), Influenza B virus (B/Kobe/1/2002), Influenza B virus (B/Kobe/1/2002-V1), Influenza B virus (B/Kobe/1/2002-V2), Influenza B virus (B/Kobe/1/2003), Influenza B virus (B/Kobe/1/94), Influenza B virus (B/Kobe/2/2002), Influenza B virus (B/Kobe/2/2003), Influenza B virus (B/Kobe/25/2003), Influenza B virus (B/Kobe/26/2003), Influenza B virus (B/Kobe/28/2003), Influenza B virus (B/Kobe/3/2002), Influenza B virus (B/Kobe/3/2003), Influenza B virus (B/Kobe/4/2002), Influenza B virus (B/Kobe/4/2003), Influenza B virus (B/Kobe/5/2002), Influenza B virus (B/Kobe/6/2002), Influenza B virus (B/Kobe/64/2001), Influenza B virus (B/Kobe/65/2001), Influenza B virus (B/Kobe/69/2001), Influenza B virus (B/Kobe/7/2002), Influenza B virus (B/Kobe/79/2001), Influenza B virus (B/Kobe/83/2001), Influenza B virus (B/Kobe/87/2001), Influenza B virus (B/Kouchi/193/1999), Influenza B virus (B/Kouchi/193/99), Influenza B virus (B/Lazio/1/02), Influenza B virus (B/Lee/40), Influenza B virus (B/Leningrad/129/91), Influenza B virus (B/Leningrad/148/91), Influenza B virus (B/Lisbon/02/1994), Influenza B virus (B/Lissabon/2/90), Influenza B virus (B/Los Angeles/1/02), Influenza B virus (B/Lusaka/270/99), Influenza B virus (B/Lusaka/432/99), Influenza B virus (B/Lyon/1271/96), Influenza B virus (B/Malaysia/83077/2001), Influenza B virus (B/Maputo/1/99), Influenza B virus (B/Maputo/2/99), Influenza B virus (B/Mar del Plata/595/99), Influenza B virus (B/Mar del Plata/VL373/99), Influenza B virus (B/Mar del Plata/VL385/99), Influenza B virus (B/Maryland/1/01), Influenza B virus (B/Maryland/1/2002), Influenza B virus (B/Maryland/2/2001), Influenza B virus (B/Maryland/7/2003), Influenza B virus (B/Massachusetts/1/2004), Influenza B virus (B/Massachusetts/2/2004), Influenza B virus (B/Massachusetts/3/2004), Influenza B virus (B/Massachusetts/4/2001), Influenza B virus (B/Massachusetts/5/2003), Influenza B virus (B/Memphis/1/01), Influenza B virus (B/Memphis/10/97), Influenza B virus (B/Memphis/11/2006), Influenza B virus (B/Memphis/12/2006), Influenza B virus (B/Memphis/12/97), Influenza B virus (B/Memphis/12/97-MA), Influenza B virus (B/Memphis/13/03), Influenza B virus (B/Memphis/18/95), Influenza B virus (B/Memphis/19/96), Influenza B virus (B/Memphis/20/96), Influenza B virus (B/Memphis/21/96), Influenza B virus (B/Memphis/28/96), Influenza B virus (B/Memphis/3/01), Influenza B virus (B/Memphis/3/89), Influenza B virus (B/Memphis/3/93), Influenza B virus (B/Memphis/4/93), Influenza B virus (B/Memphis/5/93), Influenza B virus (B/Memphis/7/03), Influenza B virus (B/Memphis/8/99), Influenza B virus (B/Mexico/84/2000), Influenza B virus (B/Michigan/04/2006), Influenza B virus (B/Michigan/1/2005), Influenza B virus (B/Michigan/1/2006), Influenza B virus (B/Michigan/2/2004), Influenza B virus (B/Michigan/20/2005), Influenza B virus (B/Michigan/22572/99), Influenza B virus (B/Michigan/22587/99), Influenza B virus (B/Michigan/22596/99), Influenza B virus (B/Michigan/22631/99), Influenza B virus (B/Michigan/22659/99), Influenza B virus (B/Michigan/22687/99), Influenza B virus (B/Michigan/22691/99), Influenza B virus (B/Michigan/22721/99), Influenza B virus (B/Michigan/22723/99), Influenza B virus (B/Michigan/2e/2006), Influenza B virus (B/Michigan/3/2004), Influenza B virus (B/Michigan/4/2006), Influenza B virus (B/Michigan/e3/2006), Influenza B virus (B/micona/1/1989), Influenza B virus (B/Mie/01/1993), Influenza B virus (B/Mie/1/93), Influenza B virus (B/Milano/1/01), Influenza B virus (B/Milano/1/02), Influenza B virus (B/Milano/5/02), Influenza B virus (B/Milano/6/02), Influenza B virus (B/Milano/66/04), Influenza B virus (B/Milano/7/02), Influenza B virus (B/Minnesota/1/1985), Influenza B virus (B/Minnesota/14/2001), Influenza B virus (B/Minnesota/2/2001), Influenza B virus (B/Minsk/318/90), Influenza B virus (B/Mississippi/1/2001), Influenza B virus (B/Mississippi/2/2005), Influenza B virus (B/Mississippi/3/2001), Influenza B virus (B/Mississippi/3/2005), Influenza B virus (B/Mississippi/4/2003), Influenza B virus (B/Mississippi/4e/2005), Influenza B virus (B/Missouri/1/2006), Influenza B virus (B/Missouri/11/2003), Influenza B virus (B/Missouri/2/2005), Influenza B virus (B/Missouri/20/2003), Influenza B virus (B/Missouri/6/2005), Influenza B virus (B/Montana/1/2003), Influenza B virus (B/Montana/1/2006), Influenza B virus (B/Montana/1e/2004), Influenza B virus (B/Moscow/16/2002), Influenza B virus (B/Moscow/3/03), Influenza B virus (B/Nagoya/20/99), Influenza B virus (B/Nairobi/2032/2006), Influenza B virus (B/Nairobi/2033/2006), Influenza B virus (B/Nairobi/2034/2006), Influenza B virus (B/Nairobi/2035/2006), Influenza B virus (B/Nairobi/351/2005), Influenza B virus (B/Nairobi/670/2005), Influenza B virus (B/Nanchang/1/00), Influenza B virus (B/Nanchang/1/2000), Influenza B virus (B/Nanchang/12/98), Influenza B virus (B/Nanchang/15/95), Influenza B virus (B/Nanchang/15/97), Influenza B virus (B/Nanchang/195/94), Influenza B virus (B/Nanchang/2/97), Influenza B virus (B/Nanchang/20/96), Influenza B virus (B/Nanchang/26/93), Influenza B virus (B/Nanchang/3/95), Influenza B virus (B/Nanchang/4/97), Influenza B virus (B/Nanchang/480/94), Influenza B virus (B/Nanchang/5/97), Influenza B virus (B/Nanchang/560/94), Influenza B virus (B/Nanchang/560a/94), Influenza B virus (B/Nanchang/560b/94), Influenza B virus (B/Nanchang/6/96), Influenza B virus (B/Nanchang/6/98), Influenza B virus (B/Nanchang/630/94), Influenza B virus (B/Nanchang/7/98), Influenza B virus (B/Nanchang/8/95), Influenza B virus (B/Nashville/107/93), Influenza B virus (B/Nashville/3/96), Influenza B virus (B/Nashville/34/96), Influenza B virus (B/Nashville/45/91), Influenza B virus (B/Nashville/48/91), Influenza B virus (B/Nashville/6/89), Influenza B virus (B/Nebraska/1/01), Influenza B virus (B/Nebraska/1/2005), Influenza B virus (B/Nebraska/2/01), Influenza B virus (B/Nebraska/4/2001), Influenza B virus (B/Nebraska/5/2003), Influenza B virus (B/Nepal/1078/2005), Influenza B virus (B/Nepal/1079/2005), Influenza B virus (B/Nepal/1080/2005), Influenza B virus (B/Nepal/1087/2005), Influenza B virus (B/Nepal/1088/2005), Influenza B virus (B/Nepal/1089/2005), Influenza B virus (B/Nepal/1090/2005), Influenza B virus (B/Nepal/1092/2005), Influenza B virus (B/Nepal/1098/2005), Influenza B virus (B/Nepal/1101/2005), Influenza B virus (B/Nepal/1103/2005), Influenza B virus (B/Nepal/1104/2005), Influenza B virus (B/Nepal/1105/2005), Influenza B virus (B/Nepal/1106/2005), Influenza B virus (B/Nepal/1108/2005), Influenza B virus (B/Nepal/1114/2005), Influenza B virus (B/Nepal/1117/2005), Influenza B virus (B/Nepal/1118/2005), Influenza B virus (B/Nepal/1120/2005), Influenza B virus (B/Nepal/1122/2005), Influenza B virus (B/Nepal/1131/2005), Influenza B virus (B/Nepal/1132/2005), Influenza B virus (B/Nepal/1136/2005), Influenza B virus (B/Nepal/1137/2005), Influenza B virus (B/Nepal/1138/2005), Influenza B virus (B/Nepal/1139/2005), Influenza B virus (B/Nepal/1331/2005), Influenza B virus (B/Netherland/2781/90), Influenza B virus (B/Netherland/6357/90), Influenza B virus (B/Netherland/800/90), Influenza B virus (B/Netherland/801/90), Influenza B virus (B/Netherlands/1/97), Influenza B virus (B/Netherlands/13/94), Influenza B virus (B/Netherlands/2/95), Influenza B virus (B/Netherlands/31/95), Influenza B virus (B/Netherlands/32/94), Influenza B virus (B/Netherlands/384/95), Influenza B virus (B/Netherlands/429/98), Influenza B virus (B/Netherlands/580/89), Influenza B virus (B/Netherlands/6/96), Influenza B virus (B/Nevada/1/2001), Influenza B virus (B/Nevada/1/2002), Influenza B virus (B/Nevada/1/2005), Influenza B virus (B/Nevada/1/2006), Influenza B virus (B/Nevada/2/2003), Influenza B virus (B/Nevada/2/2006), Influenza B virus (B/Nevada/3/2006), Influenza B virus (B/Nevada/5/2005), Influenza B virus (B/New Jersey/1/2002), Influenza B virus (B/New Jersey/1/2004), Influenza B virus (B/New Jersey/1/2005), Influenza B virus (B/New Jersey/1/2006), Influenza B virus (B/New Jersey/3/2001), Influenza B virus (B/New Jersey/3/2005), Influenza B virus (B/New Jersey/4/2001), Influenza B virus (B/New Jersey/5/2005), Influenza B virus (B/New Jersey/6/2005), Influenza B virus (B/New Mexico/1/2001), Influenza B virus (B/New Mexico/1/2006), Influenza B virus (B/New Mexico/2/2005), Influenza B virus (B/New Mexico/9/2003), Influenza B virus (B/New York/1/2001), Influenza B virus (B/New York/1/2002), Influenza B virus (B/New York/1/2004), Influenza B virus (B/New York/1/2006), Influenza B virus (B/New York/10/2002), Influenza B virus (B/New York/11/2005), Influenza B virus (B/New York/12/2001), Influenza B virus (B/New York/12/2005), Influenza B virus (B/New York/12e/2005), Influenza B virus (B/New York/14e/2005), Influenza B virus (B/New York/17/2004), Influenza B virus (B/New York/18/2003), Influenza B virus (B/New York/19/2004), Influenza B virus (B/New York/2/2000), Influenza B virus (B/New York/2/2002), Influenza B virus (B/New York/2/2006), Influenza B virus (B/New York/20139/99), Influenza B virus (B/New York/24/1993), Influenza B virus (B/New York/2e/2005), Influenza B virus (B/New York/3/90), Influenza B virus (B/New York/39/1991), Influenza B virus (B/New York/40/2002), Influenza B virus (B/New York/47/2001), Influenza B virus (B/New York/6/2004), Influenza B virus (B/New York/7/2002), Influenza B virus (B/New York/8/2000), Influenza B virus (B/New York/9/2002), Influenza B virus (B/New York/9/2004), Influenza B virus (B/New York/C10/2004), Influenza B virus (B/NIB/48/90), Influenza B virus (B/Ningxia/45/83), Influenza B virus (B/North Carolina/1/2005), Influenza B virus (B/North Carolina/3/2005), Influenza B virus (B/North Carolina/4/2004), Influenza B virus (B/North Carolina/5/2004), Influenza B virus (B/Norway/1/84), Influenza B virus (B/Ohio/1/2005), Influenza B virus (B/Ohio/1/X-19/2005), Influenza B virus (B/Ohio/1e/2005), Influenza B virus (B/Ohio/1e4/2005), Influenza B virus (B/Ohio/2/2002), Influenza B virus (B/Ohio/2e/2005), Influenza B virus (B/Oita/15/1992), Influenza B virus (B/Oklahoma/1/2006), Influenza B virus (B/Oklahoma/2/2005), Influenza B virus (B/Oman/16291/2001), Influenza B virus (B/Oman/16296/2001), Influenza B virus (B/Oman/16299/2001), Influenza B virus (B/Oman/16305/2001), Influenza B virus (B/Oregon/1/2005), Influenza B virus (B/Oregon/1/2006), Influenza B virus (B/Oregon/5/80), Influenza B virus (B/Osaka/1036/97), Influenza B virus (B/Osaka/1058/97), Influenza B virus (B/Osaka/1059/97), Influenza B virus (B/Osaka/1146/1997), Influenza B virus (B/Osaka/1169/97), Influenza B virus (B/Osaka/1201/2000), Influenza B virus (B/Osaka/547/1997), Influenza B virus (B/Osaka/547/97), Influenza B virus (B/Osaka/710/1997), Influenza B virus (B/Osaka/711/97), Influenza B virus (B/Osaka/728/1997), Influenza B virus (B/Osaka/755/1997), Influenza B virus (B/Osaka/820/1997), Influenza B virus (B/Osaka/837/1997), Influenza B virus (B/Osaka/854/1997), Influenza B virus (B/Osaka/983/1997), Influenza B virus (B/Osaka/983/1997-M1), Influenza B virus (B/Osaka/983/1997-M2), Influenza B virus (B/Osaka/983/97-V1), Influenza B virus (B/Osaka/983/97-V2), Influenza B virus (B/Osaka/983/97-V3), Influenza B virus (B/Osaka/983/97-V4), Influenza B virus (B/Osaka/983/97-V5), Influenza B virus (B/Osaka/983/97-V6), Influenza B virus (B/Osaka/983/97-V7), Influenza B virus (B/Osaka/983/97-V8), Influenza B virus (B/Osaka/c19/93), Influenza B virus (B/Oslo/1072/2001), Influenza B virus (B/Oslo/1329/2002), Influenza B virus (B/Oslo/1510/2002), Influenza B virus (B/Oslo/1846/2002), Influenza B virus (B/Oslo/1847/2002), Influenza B virus (B/Oslo/1862/2001), Influenza B virus (B/Oslo/1864/2001), Influenza B virus (B/Oslo/1870/2002), Influenza B virus (B/Oslo/1871/2002), Influenza B virus (B/Oslo/2293/2001), Influenza B virus (B/Oslo/2295/2001), Influenza B virus (B/Oslo/2297/2001), Influenza B virus (B/Oslo/238/2001), Influenza B virus (B/Oslo/3761/2000), Influenza B virus (B/Oslo/47/2001), Influenza B virus (B/Oslo/668/2002), Influenza B virus (B/Oslo/71/04), Influenza B virus (B/Oslo/801/99), Influenza B virus (B/Oslo/805/99), Influenza B virus (B/Oslo/837/99), Influenza B virus (B/Panama/45/1990), Influenza B virus (B/Panama/45/90), Influenza B virus (B/Paraguay/636/2003), Influenza B virus (B/Paris/329/90), Influenza B virus (B/Paris/549/1999), Influenza B virus (B/Parma/1/03), Influenza B virus (B/Parma/1/04), Influenza B virus (B/Parma/13/02), Influenza B virus (B/Parma/16/02), Influenza B virus (B/Parma/2/03), Influenza B virus (B/Parma/2/04), Influenza B virus (B/Parma/23/02), Influenza B virus (B/Parma/24/02), Influenza B virus (B/Parma/25/02), Influenza B virus (B/Parma/28/02), Influenza B virus (B/Parma/3/04), Influenza B virus (B/Parma/4/04), Influenza B virus (B/Parma/5/02), Influenza B virus (B/Pennsylvania/1/2006), Influenza B virus (B/Pennsylvania/2/2001), Influenza B virus (B/Pennsylvania/2/2006), Influenza B virus (B/Pennsylvania/3/2003), Influenza B virus (B/Pennsylvania/3/2006), Influenza B virus (B/Pennsylvania/4/2004), Influenza B virus (B/Perth/211/2001), Influenza B virus (B/Perth/25/2002), Influenza B virus (B/Peru/1324/2004), Influenza B virus (B/Peru/1364/2004), Influenza B virus (B/Perugia/4/03), Influenza B virus (B/Philippines/5072/2001), Influenza B virus (B/Philippines/93079/2001), Influenza B virus (B/Pusan/250/99), Influenza B virus (B/Pusan/255/99), Influenza B virus (B/Pusan/270/99), Influenza B virus (B/Pusan/285/99), Influenza B virus (B/Quebec/1/01), Influenza B virus (B/Quebec/162/98), Influenza B virus (B/Quebec/173/98), Influenza B virus (B/Quebec/2/01), Influenza B virus (B/Quebec/3/01), Influenza B virus (B/Quebec/4/01), Influenza B virus (B/Quebec/452/98), Influenza B virus (B/Quebec/453/98), Influenza B virus (B/Quebec/465/98), Influenza B virus (B/Quebec/51/98), Influenza B virus (B/Quebec/511/98), Influenza B virus (B/Quebec/514/98), Influenza B virus (B/Quebec/517/98), Influenza B virus (B/Quebec/6/01), Influenza B virus (B/Quebec/7/01), Influenza B virus (B/Quebec/74199/99), Influenza B virus (B/Quebec/74204/99), Influenza B virus (B/Quebec/74206/99), Influenza B virus (B/Quebec/8/01), Influenza B virus (B/Quebec/9/01), Influenza B virus (B/Rabat/41/97), Influenza B virus (B/Rabat/45/97), Influenza B virus (B/Rabat/61/97), Influenza B virus (B/RiodeJaneiro/200/02), Influenza B virus (B/RiodeJaneiro/209/02), Influenza B virus (B/RiodeJaneiro/315/01), Influenza B virus (B/RiodeJaneiro/353/02), Influenza B virus (B/RiodeJaneiro/354/02), Influenza B virus (B/RioGdoSul/337/01), Influenza B virus (B/RioGdoSul/357/02), Influenza B virus (B/RioGdoSul/374/01), Influenza B virus (B/Roma/1/03), Influenza B virus (B/Roma/2/03), Influenza B virus (B/Roma/3/03), Influenza B virus (B/Roma/4/02), Influenza B virus (B/Roma/7/02), Influenza B virus (B/Romania/217/1999), Influenza B virus (B/Romania/318/1998), Influenza B virus (B/Russia/22/1995), Influenza B virus (B/Saga/S172/99), Influenza B virus (B/Seal/Netherlands/1/99), Influenza B virus (B/Seoul/1/89), Influenza B virus (B/Seoul/1163/2004), Influenza B virus (B/Seoul/12/88), Influenza B virus (B/seoul/12/95), Influenza B virus (B/Seoul/13/95), Influenza B virus (B/Seoul/16/97), Influenza B virus (B/Seoul/17/95), Influenza B virus (B/Seoul/19/97), Influenza B virus (B/Seoul/21/95), Influenza B virus (B/Seoul/232/2004), Influenza B virus (B/Seoul/28/97), Influenza B virus (B/Seoul/31/97), Influenza B virus (B/Seoul/37/91), Influenza B virus (B/Seoul/38/91), Influenza B virus (B/Seoul/40/91), Influenza B virus (B/Seoul/41/91), Influenza B virus (B/Seoul/6/88), Influenza B virus (B/Shandong/7/97), Influenza B virus (B/Shangdong/7/97), Influenza B virus (B/Shanghai/1/77), Influenza B virus (B/Shanghai/10/80), Influenza B virus (B/Shanghai/24/76), Influenza B virus (B/Shanghai/35/84), Influenza B virus (B/Shanghai/361/03), Influenza B virus (B/Shanghai/361/2002), Influenza B virus (B/Shenzhen/423/99), Influenza B virus (B/Shiga/51/98), Influenza B virus (B/Shiga/N18/98), Influenza B virus (B/Shiga/T30/98), Influenza B virus (B/Shiga/T37/98), Influenza B virus (B/Shizuoka/15/2001), Influenza B virus (B/Shizuoka/480/2000), Influenza B virus (B/Sichuan/281/96), Influenza B virus (B/Sichuan/317/2001), Influenza B virus (B/Sichuan/379/99), Influenza B virus (B/Sichuan/38/2000), Influenza B virus (B/Sichuan/8/92), Influenza B virus (B/Siena/1/02), Influenza B virus (B/Singapore/04/1991), Influenza B virus (B/Singapore/11/1994), Influenza B virus (B/Singapore/22/1998), Influenza B virus (B/Singapore/222/79), Influenza B virus (B/Singapore/31/1998), Influenza B virus (B/Singapore/35/1998), Influenza B virus (B/South Australia/5/1999), Influenza B virus (B/South Carolina/04/2003), Influenza B virus (B/South Carolina/25723/99), Influenza B virus (B/South Carolina/3/2003), Influenza B virus (B/South Carolina/4/2003), Influenza B virus (B/South Dakota/1/2000), Influenza B virus (B/South Dakota/3/2003), Influenza B virus (B/South Dakota/5/89), Influenza B virus (B/Spain/WV22/2002), Influenza B virus (B/Spain/WV26/2002), Influenza B virus (B/Spain/WV27/2002), Influenza B virus (B/Spain/WV29/2002), Influenza B virus (B/Spain/WV33/2002), Influenza B virus (B/Spain/WV34/2002), Influenza B virus (B/Spain/WV36/2002), Influenza B virus (B/Spain/WV41/2002), Influenza B virus (B/Spain/WV42/2002), Influenza B virus (B/Spain/WV43/2002), Influenza B virus (B/Spain/WV45/2002), Influenza B virus (B/Spain/WV50/2002), Influenza B virus (B/Spain/WV51/2002), Influenza B virus (B/Spain/WV56/2002), Influenza B virus (B/Spain/WV57/2002), Influenza B virus (B/Spain/WV65/2002), Influenza B virus (B/Spain/WV66/2002), Influenza B virus (B/Spain/WV67/2002), Influenza B virus (B/Spain/WV69/2002), Influenza B virus (B/Spain/WV70/2002), Influenza B virus (B/Spain/WV73/2002), Influenza B virus (B/Spain/WV78/2002), Influenza B virus (B/St. Petersburg/14/2006), Influenza B virus (B/StaCatarina/308/02), Influenza B virus (B/StaCatarina/315/02), Influenza B virus (B/StaCatarina/318/02), Influenza B virus (B/StaCatarina/345/02), Influenza B virus (B/Stockholm/10/90), Influenza B virus (B/Suzuka/18/2005), Influenza B virus (B/Suzuka/28/2005), Influenza B virus (B/Suzuka/32/2005), Influenza B virus (B/Suzuka/58/2005), Influenza B virus (B/Switzerland/4291/97), Influenza B virus (B/Switzerland/5219/90), Influenza B virus (B/Switzerland/5241/90), Influenza B virus (B/Switzerland/5441/90), Influenza B virus (B/Switzerland/5444/90), Influenza B virus (B/Switzerland/5812/90), Influenza B virus (B/Switzerland/6121/90), Influenza B virus (B/Taiwan/0002/03), Influenza B virus (B/Taiwan/0114/01), Influenza B virus (B/Taiwan/0202/01), Influenza B virus (B/Taiwan/0409/00), Influenza B virus (B/Taiwan/0409/02), Influenza B virus (B/Taiwan/0562/03), Influenza B virus (B/Taiwan/0569/03), Influenza B virus (B/Taiwan/0576/03), Influenza B virus (B/Taiwan/0600/02), Influenza B virus (B/Taiwan/0610/03), Influenza B virus (B/Taiwan/0615/03), Influenza B virus (B/Taiwan/0616/03), Influenza B virus (B/Taiwan/0654/02), Influenza B virus (B/Taiwan/0684/03), Influenza B virus (B/Taiwan/0699/03), Influenza B virus (B/Taiwan/0702/02), Influenza B virus (B/Taiwan/0722/02), Influenza B virus (B/Taiwan/0730/02), Influenza B virus (B/Taiwan/0735/03), Influenza B virus (B/Taiwan/0833/03), Influenza B virus (B/Taiwan/0874/02), Influenza B virus (B/Taiwan/0879/02), Influenza B virus (B/Taiwan/0880/02), Influenza B virus (B/Taiwan/0927/02), Influenza B virus (B/Taiwan/0932/02), Influenza B virus (B/Taiwan/0993/02), Influenza B virus (B/Taiwan/1013/02), Influenza B virus (B/Taiwan/1013/03), Influenza B virus (B/Taiwan/102/2005), Influenza B virus (B/Taiwan/103/2005), Influenza B virus (B/Taiwan/110/2005), Influenza B virus (B/Taiwan/1103/2001), Influenza B virus (B/Taiwan/114/2001), Influenza B virus (B/Taiwan/11515/2001), Influenza B virus (B/Taiwan/117/2005), Influenza B virus (B/Taiwan/1197/1994), Influenza B virus (B/Taiwan/121/2005), Influenza B virus (B/Taiwan/12192/2000), Influenza B virus (B/Taiwan/1243/99), Influenza B virus (B/Taiwan/1265/2000), Influenza B virus (B/Taiwan/1293/2000), Influenza B virus (B/Taiwan/13/2004), Influenza B virus (B/Taiwan/14/2004), Influenza B virus (B/Taiwan/1484/2001), Influenza B virus (B/Taiwan/1502/02), Influenza B virus (B/Taiwan/1503/02), Influenza B virus (B/Taiwan/1534/02), Influenza B virus (B/Taiwan/1536/02), Influenza B virus (B/Taiwan/1561/02), Influenza B virus (B/Taiwan/1574/03), Influenza B virus (B/Taiwan/1584/02), Influenza B virus (B/Taiwan/16/2004), Influenza B virus (B/Taiwan/1618/03), Influenza B virus (B/Taiwan/165/2005), Influenza B virus (B/Taiwan/166/2005), Influenza B virus (B/Taiwan/188/2005), Influenza B virus (B/Taiwan/1949/02), Influenza B virus (B/Taiwan/1950/02), Influenza B virus (B/Taiwan/202/2001), Influenza B virus (B/Taiwan/2026/99), Influenza B virus (B/Taiwan/2027/99), Influenza B virus (B/Taiwan/217/97), Influenza B virus (B/Taiwan/21706/97), Influenza B virus (B/Taiwan/2195/99), Influenza B virus (B/Taiwan/2551/03), Influenza B virus (B/Taiwan/2805/01), Influenza B virus (B/Taiwan/2805/2001), Influenza B virus (B/Taiwan/3143/97), Influenza B virus (B/Taiwan/31511/00), Influenza B virus (B/Taiwan/31511/2000), Influenza B virus (B/Taiwan/34/2004), Influenza B virus (B/Taiwan/3532/03), Influenza B virus (B/Taiwan/39/2004), Influenza B virus (B/Taiwan/41010/00), Influenza B virus (B/Taiwan/41010/2000), Influenza B virus (B/Taiwan/4119/02), Influenza B virus (B/Taiwan/4184/00), Influenza B virus (B/Taiwan/4184/2000), Influenza B virus (B/Taiwan/43/2005), Influenza B virus (B/Taiwan/4602/02), Influenza B virus (B/Taiwan/473/2005), Influenza B virus (B/Taiwan/52/2004), Influenza B virus (B/Taiwan/52/2005), Influenza B virus (B/Taiwan/54/2004), Influenza B virus (B/Taiwan/61/2004), Influenza B virus (B/Taiwan/635/2005), Influenza B virus (B/Taiwan/637/2005), Influenza B virus (B/Taiwan/68/2004), Influenza B virus (B/Taiwan/68/2005), Influenza B virus (B/Taiwan/69/2004), Influenza B virus (B/Taiwan/70/2005), Influenza B virus (B/Taiwan/74/2004), Influenza B virus (B/Taiwan/75/2004), Influenza B virus (B/Taiwan/77/2005), Influenza B virus (B/Taiwan/81/2005), Influenza B virus (B/Taiwan/872/2005), Influenza B virus (B/Taiwan/97271/2001), Influenza B virus (B/Taiwan/98/2005), Influenza B virus (B/Taiwan/H96/02), Influenza B virus (B/Taiwan/M214/05), Influenza B virus (B/Taiwan/M227/05), Influenza B virus (B/Taiwan/M24/04), Influenza B virus (B/Taiwan/M244/05), Influenza B virus (B/Taiwan/M251/05), Influenza B virus (B/Taiwan/M53/05), Influenza B virus (B/Taiwan/M71/01), Influenza B virus (B/Taiwan/N1013/99), Influenza B virus (B/Taiwan/N1115/02), Influenza B virus (B/Taiwan/N1207/99), Influenza B virus (B/Taiwan/N1316/01), Influenza B virus (B/Taiwan/N1549/01), Influenza B virus (B/Taiwan/N1582/02), Influenza B virus (B/Taiwan/N16/03), Influenza B virus (B/Taiwan/N1619/04), Influenza B virus (B/Taiwan/N1848/02), Influenza B virus (B/Taiwan/N1902/04), Influenza B virus (B/Taiwan/N200/05), Influenza B virus (B/Taiwan/N2050/02), Influenza B virus (B/Taiwan/N230/01), Influenza B virus (B/Taiwan/N232/00), Influenza B virus (B/Taiwan/N2333/02), Influenza B virus (B/Taiwan/N2335/01), Influenza B virus (B/Taiwan/N253/03), Influenza B virus (B/Taiwan/N2620/04), Influenza B virus (B/Taiwan/N2986/02), Influenza B virus (B/Taiwan/N3688/04), Influenza B virus (B/Taiwan/N371/05), Influenza B virus (B/Taiwan/N376/05), Influenza B virus (B/Taiwan/N384/03), Influenza B virus (B/Taiwan/N3849/02), Influenza B virus (B/Taiwan/N404/02), Influenza B virus (B/Taiwan/N473/00), Influenza B virus (B/Taiwan/N511/01), Influenza B virus (B/Taiwan/N559/05), Influenza B virus (B/Taiwan/N612/01), Influenza B virus (B/Taiwan/N701/01), Influenza B virus (B/Taiwan/N767/01), Influenza B virus (B/Taiwan/N798/05), Influenza B virus (B/Taiwan/N860/05), Influenza B virus (B/Taiwan/N872/04), Influenza B virus (B/Taiwan/N913/04), Influenza B virus (B/Taiwan/S117/05), Influenza B virus (B/Taiwan/S141/02), Influenza B virus (B/Taiwan/S76/02), Influenza B virus (B/Taiwan/S82/02), Influenza B virus (B/Taiwn/103/2005), Influenza B virus (B/Tehran/80/02), Influenza B virus (B/Temple/B10/1999), Influenza B virus (B/Temple/B1166/2001), Influenza B virus (B/Temple/B1181/2001), Influenza B virus (B/Temple/B1182/2001), Influenza B virus (B/Temple/B1188/2001), Influenza B virus (B/Temple/B1190/2001), Influenza B virus (B/Temple/B1193/2001), Influenza B virus (B/Temple/B17/2003), Influenza B virus (B/Temple/B18/2003), Influenza B virus (B/Temple/B19/2003), Influenza B virus (B/Temple/B20/2003), Influenza B virus (B/Temple/B21/2003), Influenza B virus (B/Temple/B24/2003), Influenza B virus (B/Temple/B3/1999), Influenza B virus (B/Temple/B30/2003), Influenza B virus (B/Temple/B7/1999), Influenza B virus (B/Temple/B8/1999), Influenza B virus (B/Temple/B9/1999), Influenza B virus (B/Texas/06/2000), Influenza B virus (B/Texas/1/2000), Influenza B virus (B/Texas/1/2004), Influenza B virus (B/Texas/1/2006), Influenza B virus (B/Texas/1/91), Influenza B virus (B/Texas/10/2005), Influenza B virus (B/Texas/11/2001), Influenza B virus (B/Texas/12/2001), Influenza B virus (B/Texas/14/1991), Influenza B virus (B/Texas/14/2001), Influenza B virus (B/Texas/16/2001), Influenza B virus (B/Texas/18/2001), Influenza B virus (B/Texas/2/2006), Influenza B virus (B/Texas/22/2001), Influenza B virus (B/Texas/23/2000), Influenza B virus (B/Texas/3/2001), Influenza B virus (B/Texas/3/2002), Influenza B virus (B/Texas/3/2006), Influenza B virus (B/Texas/37/1988), Influenza B virus (B/Texas/37/88), Influenza B virus (B/Texas/4/2006), Influenza B virus (B/Texas/4/90), Influenza B virus (B/Texas/5/2002), Influenza B virus (B/Texas/57/2002), Influenza B virus (B/Texas/6/2000), Influenza B virus (B/Tokushima/101/93), Influenza B virus (B/Tokyo/6/98), Influenza B virus (B/Trento/3/02), Influenza B virus (B/Trieste/1/02), Influenza B virus (B/Trieste/1/03), Influenza B virus (B/Trieste/15/02), Influenza B virus (B/Trieste/17/02), Influenza B virus (B/Trieste/19/02), Influenza B virus (B/Trieste/2/03), Influenza B virus (B/Trieste/25/02), Influenza B virus (B/Trieste/27/02), Influenza B virus (B/Trieste/28/02), Influenza B virus (B/Trieste/34/02), Influenza B virus (B/Trieste/37/02), Influenza B virus (B/Trieste/4/02), Influenza B virus (B/Trieste/8/02), Influenza B virus (B/Trieste14/02), Influenza B virus (B/Trieste18/02), Influenza B virus (B/Trieste23/02), Influenza B virus (B/Trieste24/02), Influenza B virus (B/Trieste7/02), Influenza B virus (B/Ulan Ude/4/02), Influenza B virus (B/Ulan-Ude/6/2003), Influenza B virus (B/UlanUde/4/02), Influenza B virus (B/United Kingdom/34304/99), Influenza B virus (B/United Kingdom/34520/99), Influenza B virus (B/Uruguay/19/02), Influenza B virus (B/Uruguay/19/05), Influenza B virus (B/Uruguay/2/02), Influenza B virus (B/Uruguay/28/05), Influenza B virus (B/Uruguay/33/05), Influenza B virus (B/Uruguay/4/02), Influenza B virus (B/Uruguay/5/02), Influenza B virus (B/Uruguay/65/05), Influenza B virus (B/Uruguay/7/02), Influenza B virus (B/Uruguay/74/04), Influenza B virus (B/Uruguay/75/04), Influenza B virus (B/Uruguay/NG/02), Influenza B virus (B/Ushuaia/15732/99), Influenza B virus (B/USSR/100/83), Influenza B virus (B/Utah/1/2005), Influenza B virus (B/Utah/20139/99), Influenza B virus (B/Utah/20975/99), Influenza B virus (B/Vermont/1/2006), Influenza B virus (B/Victoria/02/1987), Influenza B virus (B/Victoria/103/89), Influenza B virus (B/Victoria/19/89), Influenza B virus (B/Victoria/2/87), Influenza B virus (B/Victoria/504/2000), Influenza B virus (B/Vienna/1/99), Influenza B virus (B/Virginia/1/2005), Influenza B virus (B/Virginia/1/2006), Influenza B virus (B/Virginia/11/2003), Influenza B virus (B/Virginia/2/2006), Influenza B virus (B/Virginia/3/2003), Influenza B virus (B/Virginia/3/2006), Influenza B virus (B/Virginia/9/2005), Influenza B virus (B/Washington/1/2004), Influenza B virus (B/Washington/2/2000), Influenza B virus (B/Washington/2/2004), Influenza B virus (B/Washington/3/2000), Influenza B virus (B/Washington/3/2003), Influenza B virus (B/Washington/5/2005), Influenza B virus (B/Wellington/01/1994), Influenza B virus (B/Wisconsin/1/2004), Influenza B virus (B/Wisconsin/1/2006), Influenza B virus (B/Wisconsin/10/2006), Influenza B virus (B/Wisconsin/15e/2005), Influenza B virus (B/Wisconsin/17/2006), Influenza B virus (B/Wisconsin/2/2004), Influenza B virus (B/Wisconsin/2/2006), Influenza B virus (B/Wisconsin/22/2006), Influenza B virus (B/Wisconsin/26/2006), Influenza B virus (B/Wisconsin/29/2006), Influenza B virus (B/Wisconsin/3/2000), Influenza B virus (B/Wisconsin/3/2004), Influenza B virus (B/Wisconsin/3/2005), Influenza B virus (B/Wisconsin/3/2006), Influenza B virus (B/Wisconsin/31/2006), Influenza B virus (B/Wisconsin/4/2006), Influenza B virus (B/Wisconsin/5/2006), Influenza B virus (B/Wisconsin/6/2006), Influenza B virus (B/Wisconsin/7/2002), Influenza B virus (B/Wuhan/2/2001), Influenza B virus (B/Wuhan/356/2000), Influenza B virus (B/WV194/2002), Influenza B virus (B/Wyoming/15/2001), Influenza B virus (B/Wyoming/16/2001), Influenza B virus (B/Wyoming/2/2003), Influenza B virus (B/Xuanwu/1/82), Influenza B virus (B/Xuanwu/23/82), Influenza B virus (B/Yamagata/1/73), Influenza B virus (B/Yamagata/115/2003), Influenza B virus (B/Yamagata/1246/2003), Influenza B virus (B/Yamagata/1311/2003), Influenza B virus (B/Yamagata/16/1988), Influenza B virus (B/Yamagata/16/88), Influenza B virus (B/Yamagata/222/2002), Influenza B virus (B/Yamagata/K198/2001), Influenza B virus (B/Yamagata/K246/2001), Influenza B virus (B/Yamagata/K270/2001), Influenza B virus (B/Yamagata/K298/2001), Influenza B virus (B/Yamagata/K320/2001), Influenza B virus (B/Yamagata/K354/2001), Influenza B virus (B/Yamagata/K386/2001), Influenza B virus (B/Yamagata/K411/2001), Influenza B virus (B/Yamagata/K461/2001), Influenza B virus (B/Yamagata/K490/2001), Influenza B virus (B/Yamagata/K500/2001), Influenza B virus (B/Yamagata/K501/2001), Influenza B virus (B/Yamagata/K508/2001), Influenza B virus (B/Yamagata/K513/2001), Influenza B virus (B/Yamagata/K515/2001), Influenza B virus (B/Yamagata/K519/2001), Influenza B virus (B/Yamagata/K520/2001), Influenza B virus (B/Yamagata/K521/2001), Influenza B virus (B/Yamagata/K535/2001), Influenza B virus (B/Yamagata/K542/2001), Influenza B virus (B/Yamanashi/166/1998), Influenza B virus (B/Yamanashi/166/98), Influenza B virus (B/Yunnan/123/2001), Influenza B virus (strain B/Alaska/12/96), Influenza B virus (STRAIN B/ANN ARBOR/1/66 [COLD-ADAPTED]), Influenza B virus (STRAIN B/ANN ARBOR/1/66 [WILD-TYPE]), Influenza B virus (STRAIN B/BA/78), Influenza B virus (STRAIN B/BEIJING/1/87), Influenza B virus (STRAIN B/ENGLAND/222/82), Influenza B virus (strain B/finland/145/90), Influenza B virus (strain B/finland/146/90), Influenza B virus (strain B/finland/147/90), Influenza B virus (strain B/finland/148/90), Influenza B virus (strain B/finland/149/90), Influenza B virus (strain B/finland/150/90), Influenza B virus (strain B/finland/151/90), Influenza B virus (strain B/finland/24/85), Influenza B virus (strain B/finland/56/88), Influenza B virus (STRAIN B/FUKUOKA/80/81), Influenza B virus (STRAIN B/GA/86), Influenza B virus (STRAIN B/GL/54), Influenza B virus (STRAIN B/HONG KONG/8/73), Influenza B virus (STRAIN B/HT/84), Influenza B virus (STRAIN B/ID/86), Influenza B virus (STRAIN B/LENINGRAD/179/86), Influenza B virus (STRAIN B/MARYLAND/59), Influenza B virus (STRAIN B/MEMPHIS/6/86), Influenza B virus (STRAIN B/NAGASAKI/1/87), Influenza B virus (strain B/Osaka/491/97), Influenza B virus (STRAIN B/PA/79), Influenza B virus (STRAIN B/RU/69), Influenza B virus (STRAIN B/SINGAPORE/64), Influenza B virus (strain B/Tokyo/942/96), Influenza B virus (STRAIN B/VICTORIA/3/85), Influenza B virus (STRAIN B/VICTORIA/87), Influenza B virus (B/Rochester/02/2001), and other subtypes. In further embodiments, the influenza virus C belongs to but is not limited to subtype Influenza C virus (C/Aichi/1/81), Influenza C virus (C/Aichi/1/99), Influenza C virus (C/Ann Arbor/1/50), Influenza C virus (C/Aomori/74), Influenza C virus (C/California/78), Influenza C virus (C/England/83), Influenza C virus (C/Fukuoka/2/2004), Influenza C virus (C/Fukuoka/3/2004), Influenza C virus (C/Fukushima/1/2004), Influenza C virus (C/Greece/79), Influenza C virus (C/Hiroshima/246/2000), Influenza C virus (C/Hiroshima/247/2000), Influenza C virus (C/Hiroshima/248/2000), Influenza C virus (C/Hiroshima/249/2000), Influenza C virus (C/Hiroshima/250/2000), Influenza C virus (C/Hiroshima/251/2000), Influenza C virus (C/Hiroshima/252/2000), Influenza C virus (C/Hiroshima/252/99), Influenza C virus (C/Hiroshima/290/99), Influenza C virus (C/Hiroshima/4/2004), Influenza C virus (C/Hyogo/1/83), Influenza C virus (C/Johannesburg/1/66), Influenza C virus (C/Johannesburg/66), Influenza C virus (C/Kanagawa/1/76), Influenza C virus (C/Kanagawa/2/2004), Influenza C virus (C/Kansas/1/79), Influenza C virus (C/Kyoto/1/79), Influenza C virus (C/Kyoto/41/82), Influenza C virus (C/Mississippi/80), Influenza C virus (C/Miyagi/1/90), Influenza C virus (C/Miyagi/1/93), Influenza C virus (C/Miyagi/1/94), Influenza C virus (C/Miyagi/1/97), Influenza C virus (C/Miyagi/1/99), Influenza C virus (C/Miyagi/12/2004), Influenza C virus (C/Miyagi/2/2000), Influenza C virus (C/Miyagi/2/92), Influenza C virus (C/Miyagi/2/93), Influenza C virus (C/Miyagi/2/94), Influenza C virus (C/Miyagi/2/96), Influenza C virus (C/Miyagi/2/98), Influenza C virus (C/Miyagi/3/2000), Influenza C virus (C/Miyagi/3/91), Influenza C virus (C/Miyagi/3/92), Influenza C virus (C/Miyagi/3/93), Influenza C virus (C/Miyagi/3/94), Influenza C virus (C/Miyagi/3/97), Influenza C virus (C/Miyagi/3/99), Influenza C virus (C/Miyagi/4/2000), Influenza C virus (C/Miyagi/4/93), Influenza C virus (C/Miyagi/4/96), Influenza C virus (C/Miyagi/4/97), Influenza C virus (C/Miyagi/4/98), Influenza C virus (C/Miyagi/42/2004), Influenza C virus (C/Miyagi/5/2000), Influenza C virus (C/Miyagi/5/91), Influenza C virus (C/Miyagi/5/93), Influenza C virus (C/Miyagi/6/93), Influenza C virus (C/Miyagi/6/96), Influenza C virus (C/Miyagi/7/91), Influenza C virus (C/Miyagi/7/93), Influenza C virus (C/Miyagi/7/96), Influenza C virus (C/Miyagi/77), Influenza C virus (C/Miyagi/8/96), Influenza C virus (C/Miyagi/9/91), Influenza C virus (C/Miyagi/9/96), Influenza C virus (C/Nara/1/85), Influenza C virus (C/Nara/2/85), Influenza C virus (C/Nara/82), Influenza C virus (C/NewJersey/76), Influenza C virus (C/Niigata/1/2004), Influenza C virus (C/Osaka/2/2004), Influenza C virus (C/pig/Beijing/115/81), Influenza C virus (C/Saitama/1/2000), Influenza C virus (C/Saitama/1/2004), Influenza C virus (C/Saitama/2/2000), Influenza C virus (C/Saitama/3/2000), Influenza C virus (C/Sapporo/71), Influenza C virus (C/Shizuoka/79), Influenza C virus (C/Yamagata/1/86), Influenza C virus (C/Yamagata/1/88), Influenza C virus (C/Yamagata/10/89), Influenza C virus (C/Yamagata/13/98), Influenza C virus (C/Yamagata/15/2004), Influenza C virus (C/Yamagata/2/2000), Influenza C virus (C/Yamagata/2/98), Influenza C virus (C/Yamagata/2/99), Influenza C virus (C/Yamagata/20/2004), Influenza C virus (C/Yamagata/20/96), Influenza C virus (C/Yamagata/21/2004), Influenza C virus (C/Yamagata/26/81), Influenza C virus (C/Yamagata/27/2004), Influenza C virus (C/Yamagata/3/2000), Influenza C virus (C/Yamagata/3/2004), Influenza C virus (C/Yamagata/3/88), Influenza C virus (C/Yamagata/3/96), Influenza C virus (C/Yamagata/4/88), Influenza C virus (C/Yamagata/4/89), Influenza C virus (C/Yamagata/5/92), Influenza C virus (C/Yamagata/6/2000), Influenza C virus (C/Yamagata/6/98), Influenza C virus (C/Yamagata/64), Influenza C virus (C/Yamagata/7/88), Influenza C virus (C/Yamagata/8/2000), Influenza C virus (C/Yamagata/8/88), Influenza C virus (C/Yamagata/8/96), Influenza C virus (C/Yamagata/9/2000), Influenza C virus (C/Yamagata/9/88), Influenza C virus (C/Yamagata/9/96), Influenza C virus (STRAIN C/BERLIN/1/85), Influenza C virus (STRAIN C/ENGLAND/892/83), Influenza C virus (STRAIN C/GREAT LAKES/1167/54), Influenza C virus (STRAIN C/JJ/50), Influenza C virus (STRAIN C/PIG/BEIJING/10/81), Influenza C virus (STRAIN C/PIG/BEIJING/439/82), Influenza C virus (STRAIN C/TAYLOR/1233/47), Influenza C virus (STRAIN C/YAMAGATA/10/81), Isavirus or Infectious salmon anemia virus, Thogotovirus or Dhori virus, Batken virus, Dhori virus (STRAIN INDIAN/1313/61) or Thogoto virus, Thogoto virus (isolate SiAr 126) or unclassified Thogotovirus, Araguari virus, unclassified Orthomyxoviridae or Fowl plague virus or Swine influenza virus or unidentified influenza virus and other subtypes.

In various embodiments, the attenuated virus belongs to the delta virus family and all related genera.

In various embodiments, the attenuated virus belongs to the Adenoviridae virus family and all related genera, strains, types and isolates for example but not limited to human adenovirus A, B C.

In various embodiments, the attenuated virus belongs to the Herpesviridae virus family and all related genera, strains, types and isolates for example but not limited to herpes simplex virus.

In various embodiments, the attenuated virus belongs to the Reoviridae virus family and all related genera, strains, types and isolates.

In various embodiments, the attenuated virus belongs to the Papillomaviridae virus family and all related genera, strains, types and isolates.

In various embodiments, the attenuated virus belongs to the Poxviridae virus family and all related genera, strains, types and isolates.

In various embodiments, the attenuated virus belongs to the Retroviridae virus family and all related genera, strains, types and isolates. For example but not limited to Human Immunodeficiency Virus.

In various embodiments, the attenuated virus belongs to the Filoviridae virus family and all related genera, strains, types and isolates.

In various embodiments, the attenuated virus belongs to the Paramyxoviridae virus family and all related genera, strains, types and isolates.

In various embodiments, the attenuated virus belongs to the Orthomyxoviridae virus family and all related genera, strains, types and isolates.

In various embodiments, the attenuated virus belongs to the Picornaviridae virus family and all related genera, strains, types and isolates.

In various embodiments, the attenuated virus belongs to the Bunyaviridae virus family and all related genera, strains, types and isolates.

In various embodiments, the attenuated virus belongs to the Nidovirales virus family and all related genera, strains, types and isolates.

In various embodiments, the attenuated virus belongs to the Caliciviridae virus family and all related genera, strains, types and isolates.

In certain embodiments, the synonymous codon substitutions alter codon bias, codon pair bias, density of deoptimized codons and deoptimized codon pairs, RNA secondary structure, CpG dinucleotide content, C+G content, translation frameshift sites, translation pause sites, the presence or absence microRNA recognition sequences or any combination thereof, in the genome. The codon substitutions may be engineered in multiple locations distributed throughout the genome, or in the multiple locations restricted to a portion of the genome. In further embodiments, the portion of the genome is the capsid coding region.

In preferred embodiments of this invention, the virus retains the ability to induce a protective immune response in an animal host. In other preferred embodiments, the virulence of the virus does not revert to wild type.

Poliovirus, Rhinovirus, and Influenza Virus

Poliovirus, a member of the Picornavirus family, is a small non-enveloped virus with a single stranded (+) sense RNA genome of 7.5 kb in length (Kitamura et al., 1981). Upon cell entry, the genomic RNA serves as an mRNA encoding a single polyprotein that after a cascade of autocatalytic cleavage events gives rise to full complement of functional poliovirus proteins. The same genomic RNA serves as a template for the synthesis of (−) sense RNA, an intermediary for the synthesis of new (+) strands that either serve as mRNA, replication template or genomic RNA destined for encapsidation into progeny virions (Mueller et al., 2005). As described herein, the well established PV system was used to address general questions of optimizing design strategies for the production of attenuated synthetic viruses. PV provides one of the most important and best understood molecular models for developing anti-viral strategies. In particular, a reverse genetics system exists whereby viral nucleic acid can be synthesized in vitro by completely synthetic methods and then converted into infectious virions (see below). Furthermore, a convenient mouse model is available (CD155tg mice, which express the human receptor for polio) for testing attenuation of synthetic PV designs as previously described (Cello et al., 2002).

Rhinoviruses are also members of the Picornavirus family, and are related to PV. Human Rhinoviruses (HRV) are the usual causative agent of the common cold, and as such they are responsible for more episodes of illness than any other infectious agent (Hendley, 1999). In addition to the common cold, HRV is also involved in ear and sinus infections, asthmatic attacks, and other diseases. Similar to PV, HRV comprises a single-stranded positive sense RNA virus, whose genome encodes a self-processing polyprotein. The RNA is translated through an internal initiation mechanism using an Internal Ribosome Entry Site (IRES) to produce structural proteins that form the capsid, as well as non-structural proteins such as the two viral proteases, 2A and 3C, and the RNA-dependent polymerase (Jang et al., 1989; Pelletier et al., 1988). Also like PV, HRV has a non-enveloped icosahedral capsid, formed by 60 copies of the four capsid proteins VP1-4 (Savolainen et al., 2003). The replication cycle of HRV is also identical to that of poliovirus. The close similarity to PV, combined with the significant, almost ubiquitous impact on human health, makes HRV an extremely attractive candidate for generating a novel attenuated virus useful for immunization.

Despite decades of research by pharmaceutical companies, no successful drug against HRV has been developed. This is partly due to the relatively low risk tolerance of federal regulators and the public for drugs that treat a mostly non-serious infection. That is, even minor side effects are unacceptable. Thus, in the absence of a drug, there is a clear desire for a safe and effective anti-rhinovirus vaccine. However, developing an anti-rhinovirus vaccine is extremely challenging, because there are over 100 serotypes of HRV, of which approximately 30 circulate widely and infect humans regularly. An effective vaccine must enable the immune system to recognize every single serotype in order to confer true immunity. The SAVE approach described herein offers a practical solution to the development of an effective rhinovirus vaccine. Based on the predictability of the SAVE design process, it would be inexpensive to design and synthesize 100 or more SAVE-attenuated rhinoviruses, which in combination would constitute a vaccine.

Influenza virus—Between 1990 and 1999, influenza viruses caused approximately 35,000 deaths each year in the U.S.A. (Thompson et al., 2003). Together with approximately 200,000 hospitalizations, the impact on the U.S. economy has been estimated to exceed $23 billion annually (Cram et al., 2001). Globally, between 300,000 to 500,000 people die each year due to influenza virus infections (Kamps et al., 2006). Although the virus causes disease amongst all age groups, the rates of serious complications are highest in children and persons over 65 years of age. Influenza has the potential to mutate or recombine into extremely deadly forms, as happened during the great influenza epidemic of 1918, in which about 30 million people died. This was possibly the single most deadly one-year epidemic in human history.

Influenza viruses are divided into three types A, B, and C. Antigenicity is determined by two glycoproteins at the surface of the enveloped virion: hemagglutinin (HA) and neuraminidase (NA). Both glycoproteins continuously change their antigenicity to escape humoral immunity. Altering the glycoproteins allows virus strains to continue infecting vaccinated individuals, which is the reason for yearly vaccination of high-risk groups. In addition, human influenza viruses can replace the HA or NA glycoproteins with those of birds and pigs, a reassortment of gene segments, known as genetic shift, leading to new viruses (H1N1 to H2N2 or H3N2, etc.) (Steinhauer and Skehel, 2002). These novel viruses, to which the global population is immunologically naive, are the cause of pandemics that kill millions of people (Kilbourne, 2006; Russell and Webster, 2005). The history of influenza virus, together with the current threat of the highly pathogenic avian influenza virus, H5N1 (Stephenson and Democratis, 2006), underscores the need for preventing influenza virus disease.

Currently, two influenza vaccines are in use: a live, attenuated vaccine (cold adapted; “FluMist”) and an inactivated virus. The application of the attenuated vaccine is restricted to healthy children, adolescents and adults (excluding pregnant females), ages 5-49. This age restriction leaves out precisely those who are at highest risks of influenza. Furthermore, the attenuated FluMist virus has the possibility of reversion, which is usual for a live virus. Production of the second, more commonly administered inactivated influenza virus vaccine is complex. Further, this vaccine appears to be less effective than hoped for in preventing death in the elderly (>65-year-old) population (Simonson et al., 2005). These facts underscore the need for novel strategies to generate influenza virus vaccines.

Reverse Genetics of Picornaviruses

Reverse genetics generally refers to experimental approaches to discovering the function of a gene that proceeds in the opposite direction to the so-called forward genetic approaches of classical genetics. That is, whereas forward genetics approaches seek to determine the function of a gene by elucidating the genetic basis of a phenotypic trait, strategies based on reverse genetics begin with an isolated gene and seek to discover its function by investigating the possible phenotypes generated by expression of the wt or mutated gene. As used herein in the context of viral systems, “reverse genetics” systems refer to the availability of techniques that permit genetic manipulation of viral genomes made of RNA. Briefly, the viral genomes are isolated from virions or from infected cells, converted to DNA (“cDNA”) by the enzyme reverse transcriptase, possibly modified as desired, and reverted, usually via the RNA intermediate, back into infectious viral particles. This process in picornaviruses is extremely simple; in fact, the first reverse genetics system developed for any animal RNA virus was for PV (Racaniello and Baltimore, 1981). Viral reverse genetics systems are based on the historical finding that naked viral genomic RNA is infectious when transfected into a suitable mammalian cell (Alexander et al., 1958). The discovery of reverse transcriptase and the development of molecular cloning techniques in the 1970's enabled scientists to generate and manipulate cDNA copies of RNA viral genomes. Most commonly, the entire cDNA copy of the genome is cloned immediately downstream of a phage T7 RNA polymerase promoter that allows the in vitro synthesis of genome RNA, which is then transfected into cells for generation of virus (van der Wert, et al., 1986). Alternatively, the same DNA plasmid may be transfected into cells expressing the T7 RNA polymerase in the cytoplasm. This system can be used for various viral pathogens including both PV and HRV.

Molecular Virology and Reverse Genetics of Influenza Virus

Influenza virus, like the picornaviruses, PV and HRV, is an RNA virus, but is otherwise unrelated to and quite different from PV. In contrast to the picornaviruses, influenza is a minus strand virus. Furthermore, influenza consists of eight separate gene segments ranging from 890 to 2341 nucleotides (Lamb and Krug, 2001). Partly because of the minus strand organization, and partly because of the eight separate gene segments, the reverse genetics system is more complex than for PV. Nevertheless, a reverse genetics system has been developed for influenza virus (Enami et al., 1990; Fodor et al., 1999; Garcia-Sastre and Palese, 1993; Hoffman et al., 2000; Luytjes et al., 1989; Neumann et al., 1999). Each of the eight gene segments is expressed from a separate plasmid. This reverse genetics system is extremely convenient for use in the SAVE strategy described herein, because the longest individual gene segment is less than 3 kb, and thus easy to synthesize and manipulate. Further, the different gene segments can be combined and recombined simply by mixing different plasmids. Thus, application of SAVE methods are possibly even more feasible for influenza virus than for PV.

A recent paradigm shift in viral reverse genetics occurred with the present inventors' first chemical synthesis of an infectious virus genome by assembly from synthetic DNA oligonucleotides (Cello et al., 2002). This achievement made it clear that most or all viruses for which a reverse genetics system is available can be synthesized solely from their genomic sequence information, and promises unprecedented flexibility in re-synthesizing and modifying these viruses to meet desired criteria.

De Novo Synthesis of Viral Genomes

Computer-based algorithms are used to design and synthesize viral genomes de novo. These synthesized genomes, exemplified by the synthesis of attenuated PV described herein, encode exactly the same proteins as wild type (wt) viruses, but by using alternative synonymous codons, various parameters, including codon bias, codon pair bias, RNA secondary structure, and/or dinucleotide content, are altered. The presented data show that these coding-independent changes produce highly attenuated viruses, often due to poor translation of proteins. By targeting an elementary function of all viruses, namely protein translation, a very general method has been developed for predictably, safely, quickly and cheaply producing attenuated viruses, which are useful for making vaccines. This method, dubbed “SAVE” (Synthetic Attenuated Virus Engineering), is applicable to a wide variety of viruses other than PV for which there is a medical need for new vaccines. These viruses include, but are not limited to rhinovirus, influenza virus, SARS and other coronaviruses, HIV, HCV, infectious bronchitis virus, ebolavirus, Marburg virus, dengue fever virus, West Nile disease virus, EBV, yellow fever virus, enteroviruses other than poliovirus, such as echoviruses, coxsackie viruses, and entrovirus71; hepatitis A virus, aphthoviruses, such as foot-and-mouth-disease virus, myxoviruses, such as influenza viruses, paramyxoviruses, such as measles virus, mumps virus, respiratory syncytia virus, flaviviruses such as dengue virus, yellow fever virus, St. Louis encephalitis virus and tick-born virus, alphaviruses, such as Western- and Eastern encephalitis virus, hepatitis B virus, and bovine diarrhea virus, and ebolavirus.

Both codon and codon-pair deoptimization in the PV capsid coding region are shown herein to dramatically reduce PV fitness. The present invention is not limited to any particular molecular mechanism underlying virus attenuation via substitution of synonymous codons. Nevertheless, experiments are ongoing to better understand the underlying molecular mechanisms of codon and codon pair deoptimization in producing attenuated viruses. In particular, evidence is provided in this application that indicates that codon deoptimization and codon pair deoptimization can result in inefficient translation. High throughput methods for the quick generation and screening of large numbers of viral constructs are also being developed.

Large-Scale DNA Assembly

In recent years, the plunging costs and increasing quality of oligonucleotide synthesis have made it practical to assemble large segments of DNA (at least up to about 10 kb) from synthetic oligonucleotides. Commercial vendors such as Blue Heron Biotechnology, Inc. (Bothwell, Wash.) (and also many others) currently synthesize, assemble, clone, sequence-verify, and deliver a large segment of synthetic DNA of known sequence for the relatively low price of about $1.50 per base. Thus, purchase of synthesized viral genomes from commercial suppliers is a convenient and cost-effective option, and prices continue to decrease rapidly. Furthermore, new methods of synthesizing and assembling very large DNA molecules at extremely low costs are emerging (Tian et al., 2004). The Church lab has pioneered a method that uses parallel synthesis of thousands of oligonucleotides (for instance, on photo-programmable microfluidics chips, or on microarrays available from Nimblegen Systems, Inc., Madison, Wis., or Agilent Technologies, Inc., Santa Clara, Calif.), followed by error reduction and assembly by overlap PCR. These methods have the potential to reduce the cost of synthetic large DNAs to less than 1 cent per base. The improved efficiency and accuracy, and rapidly declining cost, of large-scale DNA synthesis provides an impetus for the development and broad application of the SAVE strategy.

Alternative Encoding, Codon Bias, and Codon Pair Bias

Alternative Encoding

A given peptide can be encoded by a large number of nucleic acid sequences. For example, even a typical short 10-mer oligopeptide can be encoded by approximately 4¹⁰ (about 10⁶) different nucleic acids, and the proteins of PV can be encoded by about 10⁴⁴² different nucleic acids. Natural selection has ultimately chosen one of these possible 10⁴⁴² nucleic acids as the PV genome. Whereas the primary amino acid sequence is the most important level of information encoded by a given mRNA, there are additional kinds of information within different kinds of RNA sequences. These include RNA structural elements of distinct function (e.g., for PV, the cis-acting replication element, or CRE (Goodfellow et al., 2000; McKnight, 2003), translational kinetic signals (pause sites, frame shift sites, etc.), polyadenylation signals, splice signals, enzymatic functions (ribozyme) and, quite likely, other as yet unidentified information and signals).

Even with the caveat that signals such as the CRE must be preserved, 10⁴⁴² possible encoding sequences provide tremendous flexibility to make drastic changes in the RNA sequence of polio while preserving the capacity to encode the same protein. Changes can be made in codon bias or codon pair bias, and nucleic acid signals and secondary structures in the RNA can be added or removed. Additional or novel proteins can even be simultaneously encoded in alternative frames (see, e.g., Wang et al., 2006).

Codon Bias

Whereas most amino acids can be encoded by several different codons, not all codons are used equally frequently: some codons are “rare” codons, whereas others are “frequent” codons. As used herein, a “rare” codon is one of at least two synonymous codons encoding a particular amino acid that is present in an mRNA at a significantly lower frequency than the most frequently used codon for that amino acid. Thus, the rare codon may be present at about a 2-fold lower frequency than the most frequently used codon. Preferably, the rare codon is present at least a 3-fold, more preferably at least a 5-fold, lower frequency than the most frequently used codon for the amino acid. Conversely, a “frequent” codon is one of at least two synonymous codons encoding a particular amino acid that is present in an mRNA at a significantly higher frequency than the least frequently used codon for that amino acid. The frequent codon may be present at about a 2-fold, preferably at least a 3-fold, more preferably at least a 5-fold, higher frequency than the least frequently used codon for the amino acid. For example, human genes use the leucine codon CTG 40% of the time, but use the synonymous CTA only 7% of the time (see Table 2). Thus, CTG is a frequent codon, whereas CTA is a rare codon. Roughly consistent with these frequencies of usage, there are 6 copies in the genome for the gene for the tRNA recognizing CTG, whereas there are only 2 copies of the gene for the tRNA recognizing CTA. Similarly, human genes use the frequent codons TCT and TCC for serine 18% and 22% of the time, respectively, but the rare codon TCG only 5% of the time. TCT and TCC are read, via wobble, by the same tRNA, which has 10 copies of its gene in the genome, while TCG is read by a tRNA with only 4 copies. It is well known that those mRNAs that are very actively translated are strongly biased to use only the most frequent codons. This includes genes for ribosomal proteins and glycolytic enzymes. On the other hand, mRNAs for relatively non-abundant proteins may use the rare codons.

TABLE 2
Codon usage in Homo sapiens
(source: http://www.kazusa.or.jp/codon/)
Amino
Acid	Codon	Number	/1000	Fraction
Gly	GGG	636457.00	16.45	0.25
Gly	GGA	637120.00	16.47	0.25
Gly	GGT	416131.00	10.76	0.16
Gly	GGC	862557.00	22.29	0.34
Glu	GAG	1532589.00	39.61	0.58
Glu	GAA	1116000.00	28.84	0.42
Asp	GAT	842504.00	21.78	0.46
Asp	GAC	973377.00	25.16	0.54
Val	GTG	1091853.00	28.22	0.46
Val	GTA	273515.00	7.07	0.12
Val	GTT	426252.00	11.02	0.18
Val	GTC	562086.00	14.53	0.24
Ala	GCG	286975.00	7.42	0.11
Ala	GCA	614754.00	15.89	0.23
Ala	GCT	715079.00	18.48	0.27
Ala	GCC	1079491.00	27.90	0.40
Arg	AGG	461676.00	11.93	0.21
Arg	AGA	466435.00	12.06	0.21
Ser	AGT	469641.00	12.14	0.15
Ser	AGC	753597.00	19.48	0.24
Lys	AAG	1236148.00	31.95	0.57
Lys	AAA	940312.00	24.30	0.43
Asn	AAT	653566.00	16.89	0.47
Asn	AAC	739007.00	19.10	0.53
Met	ATG	853648.00	22.06	1.00
Ile	ATA	288118.00	7.45	0.17
Ile	ATT	615699.00	15.91	0.36
Ile	ATC	808306.00	20.89	0.47
Thr	ACG	234532.00	6.06	0.11
Thr	ACA	580580.00	15.01	0.28
Thr	ACT	506277.00	13.09	0.25
Thr	ACC	732313.00	18.93	0.36
Trp	TGG	510256.00	13.19	1.00
End	TGA	59528.00	1.54	0.47
Cys	TGT	407020.00	10.52	0.45
Cys	TGC	487907.00	12.61	0.55
End	TAG	30104.00	0.78	0.24
End	TAA	38222.00	0.99	0.30
Tyr	TAT	470083.00	12.15	0.44
Tyr	TAC	592163.00	15.30	0.56
Leu	TTG	498920.00	12.89	0.13
Leu	TTA	294684.00	7.62	0.08
Phe	TTT	676381.00	17.48	0.46
Phe	TTC	789374.00	20.40	0.54
Ser	TCG	171428.00	4.43	0.05
Ser	TCA	471469.00	12.19	0.15
Ser	TCT	585967.00	15.14	0.19
Ser	TCC	684663.00	17.70	0.22
Arg	CGG	443753.00	11.47	0.20
Arg	CGA	239573.00	6.19	0.11
Arg	CGT	176691.00	4.57	0.08
Arg	CGC	405748.00	10.49	0.18
Gln	CAG	1323614.00	34.21	0.74
Gln	CAA	473648.00	12.24	0.26
His	CAT	419726.00	10.85	0.42
His	CAC	583620.00	15.08	0.58
Leu	CTG	1539118.00	39.78	0.40
Leu	CTA	276799.00	7.15	0.07
Leu	CTT	508151.00	13.13	0.13
Leu	CTC	759527.00	19.63	0.20
Pro	CCG	268884.00	6.95	0.11
Pro	CCA	653281.00	16.88	0.28
Pro	CCT	676401.00	17.48	0.29
Pro	CCC	767793.00	19.84	0.32

The propensity for highly expressed genes to use frequent codons is called “codon bias.” A gene for a ribosomal protein might use only the 20 to 25 most frequent of the 61 codons, and have a high codon bias (a codon bias close to 1), while a poorly expressed gene might use all 61 codons, and have little or no codon bias (a codon bias close to 0). It is thought that the frequently used codons are codons where larger amounts of the cognate tRNA are expressed, and that use of these codons allows translation to proceed more rapidly, or more accurately, or both. The PV capsid protein is very actively translated, and has a high codon bias.

Codon Pair Bias

A distinct feature of coding sequences is their codon pair bias. This may be illustrated by considering the amino acid pair Ala-Glu, which can be encoded by 8 different codon pairs. If no factors other than the frequency of each individual codon (as shown in Table 2) are responsible for the frequency of the codon pair, the expected frequency of each of the 8 encodings can be calculated by multiplying the frequencies of the two relevant codons. For example, by this calculation the codon pair GCA-GAA would be expected to occur at a frequency of 0.097 out of all Ala-Glu coding pairs (0.23×0.42; based on the frequencies in Table 2). In order to relate the expected (hypothetical) frequency of each codon pair to the actually observed frequency in the human genome the Consensus CDS (CCDS) database of consistently annotated human coding regions, containing a total of 14,795 human genes, was used. This set of genes is the most comprehensive representation of human coding sequences. Using this set of genes the frequencies of codon usage were re-calculated by dividing the number of occurrences of a codon by the number of all synonymous codons coding for the same amino acid. As expected the frequencies correlated closely with previously published ones such as the ones given in Table 2. Slight frequency variations are possibly due to an oversampling effect in the data provided by the codon usage database at Kazusa DNA Research Institute (http://www.kazusa.or.jp/codon/codon.html) where 84949 human coding sequences were included in the calculation (far more than the actual number of human genes). The codon frequencies thus calculated were then used to calculate the expected codon-pair frequencies by first multiplying the frequencies of the two relevant codons with each other (see Table 3 expected frequency), and then multiplying this result with the observed frequency (in the entire CCDS data set) with which the amino acid pair encoded by the codon pair in question occurs. In the example of codon pair GCA-GAA, this second calculation gives an expected frequency of 0.098 (compared to 0.97 in the first calculation using the Kazusa dataset). Finally, the actual codon pair frequencies as observed in a set of 14,795 human genes was determined by counting the total number of occurrences of each codon pair in the set and dividing it by the number of all synonymous coding pairs in the set coding for the same amino acid pair (Table 3; observed frequency). Frequency and observed/expected values for the complete set of 3721 (61²) codon pairs, based on the set of 14,795 human genes, are provided herewith as Supplemental Table 1.

TABLE 3
Codon Pair Scores Exemplified
by the Amino Acid Pair Ala-Glu
amino				obs/
acid	codon	expected	observed	exp
pair	pair	frequency	frequency	ratio
AE	GCAGAA	0.098	0.163	1.65
AE	GCAGAG	0.132	0.198	1.51
AE	GCCGAA	0.171	0.031	0.18
AE	GCCGAG	0.229	0.142	0.62
AE	GCGGAA	0.046	0.027	0.57
AE	GCGGAG	0.062	0.089	1.44
AE	GCTGAA	0.112	0.145	1.29
AE	GCTGAG	0.150	0.206	1.37
Total		1.000	1.000

If the ratio of observed frequency/expected frequency of the codon pair is greater than one the codon pair is said to be overrepresented. If the ratio is smaller than one, it is said to be underrepresented. In the example the codon pair GCA-GAA is overrepresented 1.65 fold while the coding pair GCC-GAA is more than 5-fold underrepresented.

Many other codon pairs show very strong bias; some pairs are under-represented, while other pairs are over-represented. For instance, the codon pairs GCCGAA (AlaGlu) and GATCTG (AspLeu) are three- to six-fold under-represented (the preferred pairs being GCAGAG and GACCTG, respectively), while the codon pairs GCCAAG (AlaLys) and AATGAA (AsnGlu) are about two-fold over-represented. It is noteworthy that codon pair bias has nothing to do with the frequency of pairs of amino acids, nor with the frequency of individual codons. For instance, the under-represented pair GATCTG (AspLeu) happens to use the most frequent Leu codon, (CTG).

Codon pair bias was discovered in prokaryotic cells (see Greve et al., 1989), but has since been seen in all other examined species, including humans. The effect has a very high statistical significance, and is certainly not just noise. However, its functional significance, if any, is a mystery. One proposal is that some pairs of tRNAs interact well when they are brought together on the ribosome, while other pairs interact poorly. Since different codons are usually read by different tRNAs, codon pairs might be biased to avoid putting together pairs of incompatible tRNAs (Greve et al., 1989). Another idea is that many (but not all) under-represented pairs have a central CG dinucleotide (e.g., GCCGAA, encoding AlaGlu), and the CG dinucleotide is systematically under-represented in mammals (Buchan et al., 2006; Curran et al., 1995; Fedorov et al., 2002). Thus, the effects of codon pair bias could be of two kinds—one an indirect effect of the under-representation of CG in the mammalian genome, and the other having to do with the efficiency, speed and/or accuracy of translation. It is emphasized that the present invention is not limited to any particular molecular mechanism underlying codon pair bias.

As discussed more fully below, codon pair bias takes into account the score for each codon pair in a coding sequence averaged over the entire length of the coding sequence. According to the invention, codon pair bias is determined by

$\mathrm{CPB}=\sum _{i=1}^k\frac{\mathrm{CPSi}}{k-1}.$

Accordingly, similar codon pair bias for a coding sequence can be obtained, for example, by minimized codon pair scores over a subsequence or moderately diminished codon pair scores over the full length of the coding sequence.

Since all 61 sense codons and all sense codon pairs can certainly be used, it would not be expected that substituting a single rare codon for a frequent codon, or a rare codon pair for a frequent codon pair, would have much effect. Therefore, many previous investigations of codon and codon pair bias have been done via informatics, not experimentation. One investigation of codon pair bias that was based on experimental work was the study of Irwin et al. (1995), who found, counterintuitively, that certain over-represented codon pairs caused slower translation. However, this result could not be reproduced by a second group (Cheng and Goldman, 2001), and is also in conflict with results reported below. Thus, the present results (see below) may be the first experimental evidence for a functional role of codon pair bias.

Certain experiments disclosed herein relate to re-coding viral genome sequences, such as the entire capsid region of PV, involving around 1000 codons, to separately incorporate both poor codon bias and poor codon pair bias into the genome. The rationale underlying these experiments is that if each substitution creates a small effect, then all substitutions together should create a large effect. Indeed, it turns out that both deoptimized codon bias, and deoptimized codon pair bias, separately create non-viable viruses. As discussed in more detail in the Examples, preliminary data suggest that inefficient translation is the major mechanism for reducing the viability of a virus with poor codon bias or codon pair bias. Irrespective of the precise mechanism, the data indicate that the large-scale substitution of synonymous deoptimized codons into a viral genome results in severely attenuated viruses. This procedure for producing attenuated viruses has been dubbed SAVE (Synthetic Attenuated Virus Engineering).

According to the invention, viral attenuation can be accomplished by changes in codon pair bias as well as codon bias. However, it is expected that adjusting codon pair bias is particularly advantageous. For example, attenuating a virus through codon bias generally requires elimination of common codons, and so the complexity of the nucleotide sequence is reduced. In contrast, codon pair bias reduction or minimization can be accomplished while maintaining far greater sequence diversity, and consequently greater control over nucleic acid secondary structure, annealing temperature, and other physical and biochemical properties. The work disclosed herein includes attenuated codon pair bias-reduced or -minimized sequences in which codons are shuffled, but the codon usage profile is unchanged.

Viral attenuation can be confirmed in ways that are well known to one of ordinary skill in the art. Non-limiting examples induce plaque assays, growth measurements, and reduced lethality in test animals. The instant application demonstrates that the attenuated viruses are capable of inducing protective immune responses in a host.

Synthetic Attenuated Virus Engineering (SAVE)

SAVE employs specifically designed computer software and modern methods of nucleic acid synthesis and assembly to re-code and re-synthesize the genomes of viruses. This strategy provides an efficient method of producing vaccines against various medically important viruses for which efficacious vaccines are sought.

Two effective polio vaccines, an inactivated polio vaccine (IPV) developed by Jonas Salk and an oral polio vaccine (OPV) comprising live attenuated virus developed by Albert Sabin, respectively, have been available sine the 1950's. Indeed, a global effort to eradicate poliomyelitis, begun in 1988 and led by the World Health Organization (WHO), has succeeded in eradicating polio from most of the countries in the world. The number of annual diagnosed cases has been reduced from the hundreds of thousands to less that two thousand in 2005, occurring mainly in India and in Nigeria. However, a concern regarding the wide use of the OPV is that is can revert to a virulent form, and though believed to be a rare event, outbreaks of vaccine-derived polio have been reported (Georgescu et al., 1997; Kew et al., 2002; Shimizu et al., 2004). In fact, as long as the live poliovirus vaccine strains are used, each carrying less than 7 attenuating mutations, there is a possibility that this strain will revert to wt, and such reversion poses a serious threat to the complete eradication of polio. Thus, the WHO may well need a new polio vaccine to combat the potential of reversion in the closing stages of its efforts at polio eradication, and this provides one rationale for the studies disclosed herein on the application of SAVE to PV. However, PV was selected primarily because it is an excellent model system for developing SAVE.

During re-coding, essential nucleic acid signals in the viral genome are preserved, but the efficiency of protein translation is systematically reduced by deoptimizing codon bias, codon pair bias, and other parameters such as RNA secondary structure and CpG dinucleotide content, C+G content, translation frameshift sites, translation pause sites, or any combination thereof. This deoptimization may involve hundreds or thousands of changes, each with a small effect. Generally, deoptimization is performed to a point at which the virus can still be grown in some cell lines (including lines specifically engineered to be permissive for a particular virus), but where the virus is avirulent in a normal animal or human. Such avirulent viruses are excellent candidates for either a killed or live vaccine since they encode exactly the same proteins as the fully virulent virus and accordingly provoke exactly the same immune response as the fully virulent virus. In addition, the SAVE process offers the prospect for fine tuning the level of attenuation; that is, it provides the capacity to design synthetic viruses that are deoptimized to a roughly predictable extent. Design, synthesis, and production of viral particles is achievable in a timeframe of weeks once the genome sequence is known, which has important advantages for the production of vaccines in potential emergencies. Furthermore, the attenuated viruses are expected to have virtually no potential to revert to virulence because of the extremely large numbers of deleterious nucleotide changes involved. This method may be generally applicable to a wide range of viruses, requiring only knowledge of the viral genome sequence and a reverse genetics system for any particular virus.

Viral Attenuation by Deoptimizing Codon Bias

If one uses the IC₅₀-ratio of control cells/test cells method as described above, then compounds with CSG values less than or equal to 1 would not generally be considered to be good clinical candidate compounds, whereas compounds with CSG values of greater than approximately 10 would be quite promising and worthy of further consideration.

As a means of engineering attenuated viruses, the capsid coding region of poliovirus type 1 Mahoney [PV(M)] was re-engineered by making changes in synonymous codon usage. The capsid region comprises about a third of the virus and is very actively translated. One mutant virus (virus PV-AB), having a very low codon bias due to replacement of the largest possible number of frequently used codons with rare synonymous codons was created. As a control, another virus (PV-SD) was created having the largest possible number of synonymous codon changes while maintaining the original codon bias. See FIGS. 1 and 2. Thus, PV-SD is a virus having essentially the same codons as the wt, but in shuffled position while encoding exactly the same proteins. In PV-SD, no attempt was made to increase or reduce codon pair bias by the shuffling procedure. See Example 1. Despite 934 nucleotide changes in the capsid-coding region, PV-SD RNA produced virus with characteristics indistinguishable from wt. In contrast, no viable virus was recovered from PV-AB carrying 680 silent mutations. See Example 2.

A trivial explanation of the inviability of PV-AB is that just one of the nucleotide changes is somehow lethal, while the other 679 are harmless. For instance, a nucleotide change could be lethal for some catastrophic but unappreciated reason, such as preventing replication. This explanation is unlikely, however. Although PV does contain important regulatory elements in its RNA, such as the CRE, it is known that no such elements exist inside the capsid coding region. This is supported by the demonstration that the entire capsid coding region can be deleted without affecting normal replication of the residual genome within the cell, though of course viral particles cannot be formed (Kaplan and Racamiello, 1988).

To address questions concerning the inviability of certain re-engineered viruses, sub-segments of the capsid region of virus PV-AB were subcloned into the wild type virus. See Example 1 and FIG. 3. Incorporating large subcloned segments (including non-overlapping segments) proved lethal, while small subcloned segments produced viable (with one exception) but sick viruses. “Sickness” is revealed by many assays: for example, segments of poor codon bias cause poor titers (FIG. 3B) and small plaques (FIGS. 3C-H). It is particularly instructive that in general, large, lethal segments can be divided into two sub-segments, both of which are alive but sick (FIG. 3). These results rule out the hypothesis that inviability is due to just one change; instead, at minimum, many changes must be contributing to the phenotype.

There is an exceptional segment from position 1513 to 2470. This segment is fairly small, but its inclusion in the PV genome causes inviability. It is not known at present whether or not this fragment can be subdivided into subfragments that merely cause sickness and do not inactivate the virus. It is conceivable that this segment does contain a highly deleterious change, possibly a translation frameshift site.

Since poor codon bias naturally suggests an effect on translation, translation of the proteins encoded by virus PV-AB was tested. See Example 5 and FIG. 5. Indeed, all the sick viruses translated capsid protein poorly (FIG. 5B). Translation was less efficient in the sicker viruses, consistent with poor translation being the cause of the sickness. Translation was improved essentially to wt levels in reactions that were supplemented with excess tRNAs and amino acids (FIG. 5A), consistent with the rate of recognition of rare codons being limiting.

As a second test of whether deoptimized codon bias was causing inefficient translation, portions of wt and deoptimized capsid were fused to the N-terminus of firefly luciferase in a dicistronic reporter construct. See Example 5 and FIG. 6. In these fusion constructs, translation of luciferase depends on translation of the N-terminally fused capsid protein. Again, it was found that translation of the capsid proteins with deoptimized codons was poor, and was worse in the sicker viruses, suggesting a cause-and-effect relationship. Thus, the data suggest that the hundreds of rare codons in the PV-AB virus cause inviability largely because of poor translation. Further, the poor translation seen in vitro and the viral sickness seen in cultured cells are also reflected in infections of animals. Even for one of the least debilitated deoptimized viruses, PV-AB^2470-2954, the number of viral particles needed to cause disease in mice was increased by about 100-fold. See Example 4, Table 4.

Burns et al. (2006) have recently described some similar experiments with the Sabin type 2 vaccine strain of PV and reached similar conclusions. Burns et al. synthesized a completely different codon-deoptimized virus (i.e., the nucleotide sequences of the PV-AB virus described herein and their “abcd” virus are very different), and yet got a similar degree of debilitation using similar assays. Burns et al. did not test their viral constructs in host organisms for attenuation. However, their result substantiates the view that SAVE is predictable, and that the results are not greatly dependent on the exact nucleotide sequence.

Viral Attenuation by Deoptimizing Codon Pair Bias

According to the invention, codon pair bias can be altered independently of codon usage. For example, in a protein encoding sequence of interest, codon pair bias can be altered simply by directed rearrangement of its codons. In particular, the same codons that appear in the parent sequence, which can be of varying frequency in the host organism, are used in the altered sequence, but in different positions. In the simplest form, because the same codons are used as in the parent sequence, codon usage over the protein coding region being considered remains unchanged (as does the encoded amino acid sequence). Nevertheless, certain codons appear in new contexts, that is, preceded by and/or followed by codons that encode the same amino acid as in the parent sequence, but employing a different nucleotide triplet. Ideally, the rearrangement of codons results in codon pairs that are less frequent than in the parent sequence. In practice, rearranging codons often results in a less frequent codon pair at one location and a more frequent pair at a second location. By judicious rearrangement of codons, the codon pair usage bias over a given length of coding sequence can be reduced relative to the parent sequence. Alternatively, the codons could be rearranged so as to produce a sequence that makes use of codon pairs which are more frequent in the host than in the parent sequence.

Codon pair bias is evaluated by considering each codon pair in turn, scoring each pair according to the frequency that the codon pair is observed in protein coding sequences of the host, and then determining the codon pair bias for the sequence, as disclosed herein. It will be appreciated that one can create many different sequences that have the same codon pair bias. Also, codon pair bias can be altered to a greater or lesser extent, depending on the way in which codons are rearranged. The codon pair bias of a coding sequence can be altered by recoding the entire coding sequence, or by recoding one or more subsequences. As used herein, “codon pair bias” is evaluated over the length of a coding sequence, even though only a portion of the sequence may be mutated. Because codon pairs are scored in the context of codon usage of the host organism, a codon pair bias value can be assigned to wild type viral sequences and mutant viral sequences. According to the invention, a virus can be attenuated by recoding all or portions of the protein encoding sequences of the virus so a to reduce its codon pair bias.

According to the invention, codon pair bias is a quantitative property determined from codon pair usage of a host. Accordingly, absolute codon pair bias values may be determined for any given viral protein coding sequence. Alternatively, relative changes in codon pair bias values can be determined that relate a deoptimized viral protein coding sequence to a “parent” sequence from which it is derived. As viruses come in a variety of types (i.e., types I to VII by the Baltimore classification), and natural (i.e., virulent) isolates of different viruses yield different values of absolute codon pair bias, it is relative changes in codon pair bias that are usually more relevant to determining desired levels of attenuation. Accordingly, the invention provides attenuated viruses and methods of making such, wherein the attenuated viruses comprise viral genomes in which one or more protein encoding nucleotide sequences have codon pair bias reduced by mutation. In viruses that encode only a single protein (i.e., a polyprotein), all or part of the polyprotein can be mutated to a desired degree to reduce codon pair bias, and all or a portion of the mutated sequence can be provided in a recombinant viral construct. For a virus that separately encodes multiple proteins, one can reduce the codon pair bias of all of the protein encoding sequences simultaneously, or select only one or a few of the protein encoding sequences for modification. The reduction in codon pair bias is determined over the length of a protein encoding sequences, and is at least about 0.05, or at least about 0.1, or at least about 0.15, or at least about 0.2, or at least about 0.3, or at least about 0.4. Depending on the virus, the absolute codon pair bias, based on codon pair usage of the host, can be about −0.05 or less, or about −0.1 or less, or about −0.15 or less, or about −0.2 or less, or about −0.3 or less, or about −0.4 or less.

It will be apparent that codon pair bias can also be superimposed on other sequence variation. For example, a coding sequence can be altered both to encode a protein or polypeptide which contains one or more amino acid changes and also to have an altered codon pair bias. Also, in some cases, one may shuffle codons to maintain exactly the same codon usage profile in a codon-bias reduced protein encoding sequence as in a parent protein encoding sequence. This procedure highlights the power of codon pair bias changes, but need not be adhered to. Alternatively, codon selection can result in an overall change in codon usage is a coding sequence. In this regard, it is noted that in certain examples provided herein, (e.g., the design of PV-Min) even if the codon usage profile is not changed in the process of generating a codon pair bias minimized sequence, when a portion of that sequence is subcloned into an unmutated sequence (e.g., PV-MinXY or PV-MinZ), the codon usage profile over the subcloned portion, and in the hybrid produced, will not match the profile of the original unmutated protein coding sequence. However, these changes in codon usage profile have minimal effect of codon pair bias.

Similarly, it is noted that, by itself, changing a nucleotide sequence to encode a protein or polypeptide with one or many amino acid substitutions is also highly unlikely to produce a sequence with a significant change in codon pair bias. Consequently, codon pair bias alterations can be recognized even in nucleotide sequences that have been further modified to encode a mutated amino acid sequence. It is also noteworthy that mutations meant by themselves to increase codon bias are not likely to have more than a small effect on codon pair bias. For example, as disclosed herein, the codon pair bias for a poliovirus mutant recoded to maximize the use of nonpreferred codons (PV-AB) is decreased from wild type (PV-1(M)) by only about 0.05. Also noteworth is that such a protein encoding sequence have greatly diminished sequence diversity. To the contrary, substantial sequence diversity is maintained in codon pair bias modified sequences of the invention. Moreover, the significant reduction in codon pair bias obtainable without increased use of rare codons suggests that instead of maximizing the use of nonpreferred codons, as in PV-AB, it would be beneficial to rearrange nonpreferred codons with a sufficient number of preferred codons in order to more effectively reduce codon pair bias.

The extent and intensity of mutation can be varied depending on the length of the protein encoding nucleic acid, whether all or a portion can be mutated, and the desired reduction of codon pair bias. In an embodiment of the invention, a protein encoding sequence is modified over a length of at least about 100 nucleotide, or at least about 200 nucleotides, or at least about 300 nucleotides, or at least about 500 nucleotides, or at least about 1000 nucleotides.

As discussed above, the term “parent” virus or “parent” protein encoding sequence is used herein to refer to viral genomes and protein encoding sequences from which new sequences, which may be more or less attenuated, are derived. Accordingly, a parent virus can be a “wild type” or “naturally occurring” prototypes or isolate or variant or a mutant specifically created or selected on the basis of real or perceived desirable properties.

Using de novo DNA synthesis, the capsid coding region (the P1 region from nucleotide 755 to nucleotide 3385) of PV(M) was redesigned to introduce the largest possible number of rarely used codon pairs (virus PV-Min) (SEQ ID NO:4) or the largest possible number of frequently used codon pairs (virus PV-Max) (SEQ ID NO:5), while preserving the codon bias of the wild type virus. See Example 7. That is, the designed sequences use the same codons as the parent sequence, but they appear in a different order. The PV-Max virus exhibited one-step growth kinetics and killing of infected cells essentially identical to wild type virus. (That growth kinetics are not increased for a codon pair maximized virus relative to wild type appears to hold true for other viruses as well.) Conversely, cells transfected with PV-Min mutant RNA were not killed, and no viable virus could be recovered. Subcloning of fragments (PV-Min^755-2470, PV-Min^2470-3386) of the capsid region of PV-Min into the wt background produced very debilitated, but not dead, virus. See Example 7 and FIG. 8. This result substantiates the hypothesis that deleterious codon changes are preferably widely distributed and demonstrates the simplicity and effectiveness of varying the extent of the codon pair deoptimized sequence that is substituted into a wild type parent virus genome in order to vary the codon pair bias for the overall sequence and the attenuation of the viral product. As seen with PV-AB viruses, the phenotype of PV-Min viruses is a result of reduced specific infectivity of the viral particles rather than of lower production of progeny virus.

Virus with deoptimized codon pair bias are attenuated. As exemplified below, (see Example 8, and Table 5), CD155tg mice survived challenge by intracerebral injection of attenuated virus in amounts 1000-fold higher than would be lethal for wild type virus. These findings demonstrate the power of deoptimization of codon pair bias to minimize lethality of a virus. Further, the viability of the virus can be balanced with a reduction of infectivity by choosing the degree of codon pair bias deoptimization. Further, once a degree or ranges of degrees of codon pair bias deoptimization is determined that provides desired attenuation properties, additional sequences can be designed to attain that degree of codon pair bias. For example, SEQ ID NO:6 provides a poliovirus sequence with a codon pair bias of about −0.2, and mutations distributed over the region encompassing the mutated portions of PV-MinXY and PV-MinZ (i.e., PV^755-3385).

Algorithms for Sequence Design

The inventors have developed several novel algorithms for gene design that optimize the DNA sequence for particular desired properties while simultaneously coding for the given amino acid sequence. In particular, algorithms for maximizing or minimizing the desired RNA secondary structure in the sequence (Cohen and Skiena, 2003) as well as maximally adding and/or removing specified sets of patterns (Skiena, 2001), have been developed. The former issue arises in designing viable viruses, while the latter is useful to optimally insert restriction sites for technological reasons. The extent to which overlapping genes can be designed that simultaneously encode two or more genes in alternate reading frames has also been studied (Wang et al., 2006). This property of different functional polypeptides being encoded in different reading frames of a single nucleic acid is common in viruses and can be exploited for technological purposes such as weaving in antibiotic resistance genes.

The first generation of design tools for synthetic biology has been built, as described by Jayaraj et al. (2005) and Richardson et al. (2006). These focus primarily on optimizing designs for manufacturability (i.e., oligonucleotides without local secondary structures and end repeats) instead of optimizing sequences for biological activity. These first-generation tools may be viewed as analogous to the early VLSI CAD tools built around design rule-checking, instead of supporting higher-order design principles.

As exemplified herein, a computer-based algorithm can be used to manipulate the codon pair bias of any coding region. The algorithm has the ability to shuffle existing codons and to evaluate the resulting CPB, and then to reshuffle the sequence, optionally locking in particularly “valuable” codon pairs. The algorithm also employs a for of “simulated annealing” so as not to get stuck in local minima Other parameters, such as the free energy of folding of RNA, may optional be under the control of the algorithm as well, in order to avoid creation of undesired secondary structures. The algorithm can be used to find a sequence with a minimum codon pair bias, and in the event that such a sequence does not provide a viable virus, the algorithm can be adjusted to find sequences with reduced, but not minimized biases. Of course, a viable viral sequence could also be produced using only a subsequence of the computer minimized sequence.

Whether or not performed with the aid of a computer, using, for example, a gradient descent, or simulated annealing, or other minimization routine. An example of the procedure that rearranges codons present in a starting sequence can be represented by the following steps:

1) Obtain wildtype viral genome sequence.

2) Select protein coding sequences to target for attenuated design.

3) Lock down known or conjectured DNA segments with non-coding functions.

4) Select desired codon distribution for remaining amino acids in redesigned proteins.

5) Perform random shuffle of unlocked codon positions and calculate codon-pair score.

6) Further reduce (or increase) codon-pair score optionally employing a simulated annealing procedure.

7) Inspect resulting design for excessive secondary structure and unwanted restriction site:

• • if yes→go to step (5) or correct the design by replacing problematic regions with wildtype sequences and go to step (8).

8. Synthesize DNA sequence corresponding to virus design.

9. Create viral construct and assess expression:

• • if too attenuated, prepare subclone construct and goto 9;
  • if insufficiently attenuated, goto 2.

Source code (PERL script) of a computer based simulated annealing routine is provided.

Alternatively, one can devise a procedure which allows each pair of amino acids to be deoptimized by choosing a codon pair without a requirement that the codons be swapped out from elsewhere in the protein encoding sequence.

Molecular Mechanisms of Viral Attenuation: Characterization of Attenuated PV Using High-Throughput Methods

As described above and in greater detail in the Examples, two synthetic, attenuated polioviruses encoding exactly the same proteins as the wildtype virus, but having altered codon bias or altered codon pair bias, were constructed. One virus uses deoptimized codons; the other virus uses deoptimized codon pairs. Each virus has many hundreds of nucleotide changes with respect to the wt virus.

The data presented herein suggest that these viruses are attenuated because of poor translation. This finding, if correct, has important consequences. First, the reduced fitness/virulence of each virus is due to small defects at hundreds of positions spread over the genome. Thus, there is essentially no chance of the virus reverting to wildtype, and so the virus is a good starting point for either a live or killed vaccine. Second, if the reduced fitness/virulence is due to additive effects of hundreds of small defects in translation, this method of reducing fitness with minimal risk of reversion should be applicable to many other viruses.

Though it is emphasized that the present invention is not limited to any particular mode of operation or underlying molecular mechanism, ongoing studies are aimed at distinguishing these alternative hypotheses. The ongoing investigations involve use of high throughput methods to scan through the genomes of various attenuated virus designs such as codon and codon pair deoptimized poliovirus and influenza virus, and to construct chimeras by placing overlapping 300-bp portions of each mutant virus into a wt context. See Example 11. The function of these chimeric viruses are then assayed. A finding that most chimeras are slightly, but not drastically, less fit than wild type, as suggested by the preliminary data disclosed herein, corroborates the “incremental loss of function” hypothesis, wherein many deleterious mutations are distributed throughout the regions covered by the chimeras. Conversely, a finding that most of the chimeras are similar or identical to wt, whereas one or only a few chimeras are attenuated like the parental mutant, suggests that there are relatively few positions in the sequence where mutation results in attenuation and that attenuation at those positions is significant.

As described in Example 12, experiments are performed to determine how codon and codon-pair deoptimization affect RNA stability and abundance, and to pinpoint the parameters that impair translation of the re-engineered viral genome. An understanding of the molecular basis of this impairment will further enhance the applicability of the SAVE approach to a broad range of viruses. Another conceivable mechanism underlying translation impairment is translational frameshifting, wherein the ribosome begins to translate a different reading frame, generating a spurious, typically truncated polypeptide up to the point where it encounters an in-frame stop codon. The PV genomes carrying the AB mutant segment from residue 1513 to 2470 are not only non-viable, but also produce a novel protein band during in vitro translation of approximately 42-44 kDa (see FIG. 5A). The ability of this AB^1513-2470 fragment to inactivate PV, as well as its ability to induce production of the novel protein, may reflect the occurrence of a frameshift event and this possibility is also being investigated. A filter for avoiding the introduction of frameshifting sites is built into the SAVE design software.

More detailed investigations of translational defects are conducted using various techniques including, but not limited to, polysome profiling, toeprinting, and luciferase assays of fusion proteins, as described in Example 12.

Molecular Biology of Poliovirus

While studies are ongoing to unravel the mechanisms underlying viral attenuation by SAVE, large-scale codon deoptimization of the PV capsid coding region revealed interesting insights into the biology of PV itself. What determines the PFU/particle ratio (specific infectivity) of a virus has been a longstanding question. In general, failure at any step during the infectious life cycle before the establishment of a productive infection will lead to an abortive infection and, therefore, to the demise of the infecting particle. In the case of PV, it has been shown that approximately 100 virions are required to result in one infectious event in cultured cells (Joklik and Darnell, 1961; Schwerdt and Fogh, 1957). That is, of 100 particles inoculated, only approximately one is likely to successfully complete all steps at the level of receptor binding (step 1), followed by internalization and uncoating (step 2), initiation of genome translation (step 3), polyprotein translation (step 4), RNA replication (step 5), and encapsidation of progeny (step 6).

In the infectious cycle of AB-type viruses described here, steps 1 and 2 should be identical to a PV(M) infection as their capsids are identical. Likewise, identical 5′ nontranslated regions should perform equally well in assembly of a translation complex (step 3). Viral polyprotein translation, on the other hand (step 4), is severely debilitated due to the introduction of a great number of suboptimal synonymous codons in the capsid region (FIGS. 5 and 6). It is thought that the repeated encounter of rare codons by the translational machinery causes stalling of the ribosome as, by the laws of mass action, rare aminoacyl-tRNA will take longer to diffuse into the A site on the ribosome. As peptide elongation to a large extent is driven by the concentration of available aminoacyl-tRNA, dependence of an mRNA on many rare tRNAs consequently lengthens the time of translation (Gustafsson et al., 2004). Alternatively, excessive stalling of the ribosome may cause premature dissociation of the translation complex from the RNA and result in a truncated protein destined for degradation. Both processes lead to a lower protein synthesis rate per mRNA molecule per unit of time. While the data presented herein suggest that the phenotypes of codon-deoptimized viruses are determined by the rate of genome translation, other mechanistic explanations may be possible. For example, it has been suggested that the conserved positions of rare synonymous codons throughout the viral capsid sequence in Hepatitis A virus are of functional importance for the proper folding of the nascent polypeptide by introducing necessary translation pauses (Sanchez et al., 2003). Accordingly, large-scale alteration of the codon composition may conceivably change some of these pause sites to result in an increase of misfolded capsid proteins.

Whether these considerations also apply to the PV capsid is not clear. If so, an altered phenotype would have been expected with the PV-SD design, in which the wt codons were preserved, but their positions throughout the capsid were completely changed. That is, none of the purported pause sites would be at the appropriate position with respect to the protein sequence. No change in phenotype, however, was observed and PV-SD translated and replicated at wild type levels (FIG. 3B).

Another possibility is that the large-scale codon alterations in the tested designs may create fortuitous dominant-negative RNA elements, such as stable secondary structures, or sequences that may undergo disruptive long-range interactions with other regions of the genome.

It is assumed that all steps prior to, and including, virus uncoating should be unchanged when wt and the mutant viruses, described herein are compared. This is supported by the observation that the eclipse period for all these isolates is similar (FIG. 3B). The dramatic reduction in PFU/particle ratio is, therefore, likely to be a result of the reduced translation capacity of the deoptimized genomes, i.e., the handicap of the mutant viruses is determined intracellularly.

It is generally assumed that the relatively low PFU/particle ratio of picornaviruses of 1/100 to 1/1,000 (Rueckert, 1985) is mainly determined by structural alterations at the receptor binding step, either prior to or at the level of cell entry. The formation of 135S particles that are hardly infectious may be the major culprit behind the inefficiency of poliovirus infectivity (Hogle, 2002). However, certain virus mutants seem to sidestep A particle conversion without resulting in a higher specific infectivity, an observation suggesting that other post-entry mechanisms may be responsible for the low PFU/particle ratio (Dove and Racaniello, 1997).

The present data provide clear evidence for such post-entry interactions between virus and cell, and suggest that these, and not pre-entry events, contribute to the distinct PFU/particle ratio of poliovirus. As all replication proteins in poliovirus are located downstream of P1 on the polyprotein, they critically depend upon successful completion of P1 translation. Lowering the rate of P1 translation therefore lowers translation of all replication proteins to the same extent. This, in turn, likely leads to a reduced capacity of the virus to make the necessary modifications to the host cell required for establishment of a productive infection, such as shutdown of host cell translation or prevention of host cell innate responses. While codon deoptimization, as described herein, is likely to effect translation at the peptide elongation step, reduced initiation of translation can also be a powerful attenuating determinant as well, as has been shown for mutations in the internal ribosomal entry site in the Sabin vaccine strains of poliovirus (Svitkin et al., 1993; 1985).

On the basis of these considerations, it is predicted that many mutant phenotypes attributable to defects in genome translation or early genome replication actually manifest themselves by lowering PFU/particle ratios. This would be the case as long as the defect results in an increased chance of abortive infection. Since in almost all studies the omnipresent plaque assay is the virus detection method of choice, a reduction in the apparent virus titer is often equated with a reduction in virus production per se. This may be an inherent pitfall that can be excused with the difficulties of characterizing virus properties at the single-cell level. Instead, most assays are done on a large population of cells. A lower readout of the chosen test (protein synthesis, RNA replication, virus production as measured in PFU) is taken at face value as an indicator of lower production on a per-cell basis, without considering that virus production in a cell may be normal while the number of cells producing virus is reduced.

The near-identical production of particles per cell by codon-deoptimized viruses indicates that the total of protein produced after extended period of times is not severely affected, whereas the rate of protein production has been drastically reduced. This is reflected in the delayed appearance of CPE, which may be a sign that the virus has to go through more RNA replication cycles to build up similar intracellular virus protein concentrations. It appears that codon-deoptimized viruses are severely handicapped in establishing a productive infection because the early translation rate of the incoming infecting genome is reduced. As a result of this lower translation rate, PV proteins essential for disabling the cell's antiviral responses (most likely proteinases 2A^pro and 3C^pro) are not synthesized at sufficient amounts to pass this crucial hurdle in the life cycle quickly enough. Consequently, there is a better chance for the cell to eliminate the infection before viral replication could unfold and take over the cell. Thus, the likelihood for productive infection events is reduced and the rate of abortive infection is increased. However, in the case where a codon-deoptimized virus does succeed in disabling the cell, this virus will produce nearly identical amounts of progeny to the wild type. The present data suggest that a fundamental difference may exist between early translation (from the incoming RNA genome) and late translation during the replicative phase, when the cell's own translation is largely shut down. Although this may be a general phenomenon, it might be especially important in the case of codon-deoptimized genomes. Host cell shutoff very likely results in an over-abundance of free aminoacyl-tRNAs, which may overcome the imposed effect of the suboptimal codon usage as the PV genomes no longer have to compete with cellular RNAs for translation resources. This, in fact, may be analogous to observations with the modified in vitro translation system described herein (FIG. 5B). Using a translation extract that was not nuclease-treated (and thus contained cellular mRNAs) and not supplemented with exogenous amino acids or tRNAs, clear differences were observed in the translation capacity of different capsid design mutants. Under these conditions, viral genomes have to compete with cellular mRNAs in an environment where supplies are limited. In contrast, in the traditional translation extract, in which endogenous mRNAs were removed and excess tRNAs and amino acids were added, all PV RNAs translated equally well regardless of codon bias (FIG. 5A). These two different in vitro conditions may be analogous to in vivo translation during the early and late phases in the PV-infected cell.

One key finding of the present study is the realization that, besides the steps during the physical interaction and uptake of virus, the PFU/particle ratio also largely reflects the virus' capacity to overcome host cell antiviral responses. This suggests that picornaviruses are actually quite inefficient in winning this struggle, and appear to have taken the path of evolving small genomes that can quickly replicate before the cell can effectively respond. As the data show, slowing down translation rates by only 30% in PV-AB^2470-2954 (see FIG. 6) leads to a 1,000-fold higher rate of abortive infection as reflected in the lower specific infectivity (FIG. 4D). Picornaviruses apparently not only replicate at the threshold of error catastrophe (Crotty et al., 2001; Holland et al., 1990) but also at the threshold of elimination by the host cell's antiviral defenses. This effect may have profound consequences for the pathogenic phenotype of a picornavirus. The cellular antiviral processes responsible for the increased rate of aborted infections by codon-deoptimized viruses are not completely understood at present. PV has been shown to both induce and inhibit apoptosis (Belov et al., 2003; Girard et al., 1999; Tolskaya et al., 1995). Similarly PV interferes with the interferon pathway by cleaving NF-κB (Neznanov et al., 2005). It is plausible that a PV with a reduced rate of early genome translation still induces antiviral responses in the same way as a wt virus (induction of apoptosis and interferon by default) but then, due to low protein synthesis, has a reduced potential of inhibiting these processes. This scenario would increase the likelihood of the cell aborting a nascent infection and could explain the observed phenomena. At the individual cell level, PV infection is likely to be an all-or-nothing phenomenon. Viral protein and RNA syntheses likely need to be within a very close to maximal range in order to ensure productive infection.

Attenuated Virus Vaccine Compositions

The present invention provides a vaccine composition for inducing a protective immune response in a subject comprising any of the attenuated viruses described herein and a pharmaceutically acceptable carrier.

It should be understood that an attenuated virus of the invention, where used to elicit a protective immune response in a subject or to prevent a subject from becoming afflicted with a virus-associated disease, is administered to the subject in the form of a composition additionally comprising a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known to those skilled in the art and include, but are not limited to, one or more of 0.01-0.1M and preferably 0.05M phosphate buffer, phosphate-buffered saline (PBS), or 0.9% saline. Such carriers also include aqueous or non-aqueous solutions, suspensions, and emulsions. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, saline and buffered media. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's and fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Solid compositions may comprise nontoxic solid carriers such as, for example, glucose, sucrose, mannitol, sorbitol, lactose, starch, magnesium stearate, cellulose or cellulose derivatives, sodium carbonate and magnesium carbonate. For administration in an aerosol, such as for pulmonary and/or intranasal delivery, an agent or composition is preferably formulated with a nontoxic surfactant, for example, esters or partial esters of C6 to C22 fatty acids or natural glycerides, and a propellant. Additional carriers such as lecithin may be included to facilitate intranasal delivery. Pharmaceutically acceptable carriers can further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives and other additives, such as, for example, antimicrobials, antioxidants and chelating agents, which enhance the shelf life and/or effectiveness of the active ingredients. The instant compositions can, as is well known in the art, be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to a subject.

In various embodiments of the instant vaccine composition, the attenuated virus (i) does not substantially alter the synthesis and processing of viral proteins in an infected cell; (ii) produces similar amounts of virions per infected cell as wt virus; and/or (iii) exhibits substantially lower virion-specific infectivity than wt virus. In further embodiments, the attenuated virus induces a substantially similar immune response in a host animal as the corresponding wt virus.

This invention also provides a modified host cell line specially isolated or engineered to be permissive for an attenuated virus that is inviable in a wild type host cell. Since the attenuated virus cannot grow in normal (wild type) host cells, it is absolutely dependent on the specific helper cell line for growth. This provides a very high level of safety for the generation of virus for vaccine production. Various embodiments of the instant modified cell line permit the growth of an attenuated virus, wherein the genome of said cell line has been altered to increase the number of genes encoding rare tRNAs.

In preferred embodiments, the rare codons are CTA (coding for Leu), TCG (Ser), and CCG (Pro). In different embodiments, one, two, or all three of these rare codons are substituted for synonymous frequent codons in the viral genome. For example, all Leu codons in the virus may be changed to CTA; all Ser codons may be changed to TCG; all Pro codons may be changed to CCG; the Leu and Ser, or Leu and Pro, or Ser and Pro codons may be replaced by the identified rare codons; or all Leu, Ser, and Pro codons may be changed to CTA, TCG, and CCG, respectively, in a single virus. Further, a fraction of the relevant codons, i.e., less than 100%, may be changed to the rare codons. Thus, the proportion of codons substituted may be about 20%, 40%, 60%, 80% or 100% of the total number of codons.

In certain embodiments, these substitutions are made only in the capsid region of the virus, where a high rate of translation is most important. In other embodiments, the substitutions are made throughout the virus. In further embodiments, the cell line over-expresses tRNAs that bind to the rare codons.

This invention further provides a method of synthesizing any of the attenuated viruses described herein, the method comprising (a) identifying codons in multiple locations within at least one non-regulatory portion of the viral genome, which codons can be replaced by synonymous codons; (b) selecting a synonymous codon to be substituted for each of the identified codons; and (c) substituting a synonymous codon for each of the identified codons.

In certain embodiments of the instant methods, steps (a) and (b) are guided by a computer-based algorithm for Synthetic Attenuated Virus Engineering (SAVE) that permits design of a viral genome by varying specified pattern sets of deoptimized codon distribution and/or deoptimized codon-pair distribution within preferred limits. The invention also provides a method wherein, the pattern sets alternatively or additionally comprise, density of deoptimized codons and deoptimized codon pairs, RNA secondary structure, CpG dinucleotide content, C+G content, overlapping coding frames, restriction site distribution, frameshift sites, or any combination thereof.

In other embodiments, step (c) is achieved by de novo synthesis of DNA containing the synonymous codons and/or codon pairs and substitution of the corresponding region of the genome with the synthesized DNA. In further embodiments, the entire genome is substituted with the synthesized DNA. In still further embodiments, a portion of the genome is substituted with the synthesized DNA. In yet other embodiments, said portion of the genome is the capsid coding region.

In addition, the present invention provides a method for eliciting a protective immune response in a subject comprising administering to the subject a prophylactically or therapeutically effective dose of any of the vaccine compositions described herein. This invention also provides a method for preventing a subject from becoming afflicted with a virus-associated disease comprising administering to the subject a prophylactically effective dose of any of the instant vaccine compositions. In embodiments of the above methods, the subject has been exposed to a pathogenic virus. “Exposed” to a pathogenic virus means contact with the virus such that infection could result.

The invention further provides a method for delaying the onset, or slowing the rate of progression, of a virus-associated disease in a virus-infected subject comprising administering to the subject a therapeutically effective dose of any of the instant vaccine compositions.

As used herein, “administering” means delivering using any of the various methods and delivery systems known to those skilled in the art. Administering can be performed, for example, intraperitoneally, intracerebrally, intravenously, orally, transmucosally, subcutaneously, transdermally, intradermally, intramuscularly, topically, parenterally, via implant, intrathecally, intralymphatically, intralesionally, pericardially, or epidurally. An agent or composition may also be administered in an aerosol, such as for pulmonary and/or intranasal delivery. Administering may be performed, for example, once, a plurality of times, and/or over one or more extended periods.

Eliciting a protective immune response in a subject can be accomplished, for example, by administering a primary dose of a vaccine to a subject, followed after a suitable period of time by one or more subsequent administrations of the vaccine. A suitable period of time between administrations of the vaccine may readily be determined by one skilled in the art, and is usually on the order of several weeks to months. The present invention is not limited, however, to any particular method, route or frequency of administration.

A “subject” means any animal or artificially modified animal. Animals include, but are not limited to, humans, non-human primates, cows, horses, sheep, pigs, dogs, cats, rabbits, ferrets, rodents such as mice, rats and guinea pigs, and birds. Artificially modified animals include, but are not limited to, SCID mice with human immune systems, and CD155tg transgenic mice expressing the human poliovirus receptor CD155. In a preferred embodiment, the subject is a human. Preferred embodiments of birds are domesticated poultry species, including, but not limited to, chickens, turkeys, ducks, and geese.

A “prophylactically effective dose” is any amount of a vaccine that, when administered to a subject prone to viral infection or prone to affliction with a virus-associated disorder, induces in the subject an immune response that protects the subject from becoming infected by the virus or afflicted with the disorder. “Protecting” the subject means either reducing the likelihood of the subject's becoming infected with the virus, or lessening the likelihood of the disorder's onset in the subject, by at least two-fold, preferably at least ten-fold. For example, if a subject has a 1% chance of becoming infected with a virus, a two-fold reduction in the likelihood of the subject becoming infected with the virus would result in the subject having a 0.5% chance of becoming infected with the virus. Most preferably, a “prophylactically effective dose” induces in the subject an immune response that completely prevents the subject from becoming infected by the virus or prevents the onset of the disorder in the subject entirely.

As used herein, a “therapeutically effective dose” is any amount of a vaccine that, when administered to a subject afflicted with a disorder against which the vaccine is effective, induces in the subject an immune response that causes the subject to experience a reduction, remission or regression of the disorder and/or its symptoms. In preferred embodiments, recurrence of the disorder and/or its symptoms is prevented. In other preferred embodiments, the subject is cured of the disorder and/or its symptoms.

Certain embodiments of any of the instant immunization and therapeutic methods further comprise administering to the subject at least one adjuvant. An “adjuvant” shall mean any agent suitable for enhancing the immunogenicity of an antigen and boosting an immune response in a subject. Numerous adjuvants, including particulate adjuvants, suitable for use with both protein- and nucleic acid-based vaccines, and methods of combining adjuvants with antigens, are well known to those skilled in the art. Suitable adjuvants for nucleic acid based vaccines include, but are not limited to, Quil A, imiquimod, resiquimod, and interleukin-12 delivered in purified protein or nucleic acid form. Adjuvants suitable for use with protein immunization include, but are not limited to, alum, Freund's incomplete adjuvant (FIA), saponin, Quil A, and QS-21.

The invention also provides a kit for immunization of a subject with an attenuated virus of the invention. The kit comprises the attenuated virus, a pharmaceutically acceptable carrier, an applicator, and an instructional material for the use thereof. In further embodiments, the attenuated virus may be one or more poliovirus, one or more rhinovirus, one or more influenza virus, etc. More than one virus may be preferred where it is desirable to immunize a host against a number of different isolates of a particular virus. The invention includes other embodiments of kits that are known to those skilled in the art. The instructions can provide any information that is useful for directing the administration of the attenuated viruses.

Of course, it is to be understood and expected that variations in the principles of invention herein disclosed can be made by one skilled in the art and it is intended that such modifications are to be included within the scope of the present invention. The following Examples further illustrate the invention, but should not be construed to limit the scope of the invention in any way. Detailed descriptions of conventional methods, such as those employed in the construction of recombinant plasmids, transfection of host cells with viral constructs, polymerase chain reaction (PCR), and immunological techniques can be obtained from numerous publications, including Sambrook et al. (1989) and Coligan et al. (1994). All references mentioned herein are incorporated in their entirety by reference into this application.

Full details for the various publications cited throughout this application are provided at the end of the specification immediately preceding the claims. The disclosures of these publications are hereby incorporated in their entireties by reference into this application. However, the citation of a reference herein should not be construed as an acknowledgement that such reference is prior art to the present invention.

Example 1

Re-Engineering of Capsid Region of Polioviruses by Altering Codon Bias

Cells, Viruses, Plasmids, and Bacteria

HeLa R19 cell monolayers were maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% bovine calf serum (BCS) at 37° C. All PV infectious cDNA constructs are based on PV1(M) cDNA clone pT7PVM (Cao et al., 1993; van der Werf et al., 1986). Dicistronic reporter plasmids were constructed using pHRPF-Luc (Zhao and Wimmer, 2001). Escherichia coli DH5α was used for plasmid transformation and propagation. Viruses were amplified by infection of HeLa R19 cell monolayers with 5 PFU per cell. Infected cells were incubated in DMEM (2% BCS) at 37° C. until complete cytopathic effect (CPE) was apparent or for at least 4 days post-infection. After three rounds of freezing and thawing, the lysate was clarified of cell debris by low-speed centrifugation and the supernatant, containing the virus, was used for further passaging or analysis.

Cloning of Synthetic Capsid Replacements and Dicistronic Reporter Replicons

Two PV genome cDNA fragments spanning the genome between nucleotides 495 and 3636, named SD and AB, were synthesized using GeneMaker® technology (Blue Heron Biotechnology). pPV-SD and pPV-AB were generated by releasing the replacement cassettes from the vendor's cloning vector by PflMI digestion and insertion into the pT7PVM vector in which the corresponding PflMI fragment had been removed. pPV-AB^755-1513 and pPV-AB^2470-3386 were obtained by inserting a BsmI fragment or an NheI-EcoRI fragment, respectively, from pPV-AB into equally digested pT7PVM vector. In pPV-AB^1513-3386 and pPV-AB^755-2479, the BsmI fragment or NheI-EcoRI fragment of pT7PVM, respectively, replaces the respective fragment of the pPV-AB vector. Replacement of the NheI-EcoRI fragment of pPV-AB^1513-3386 with that of pT7PVM resulted in pPV-AB^2470-3386. Finally, replacement of the SnaBI-EcoRI fragments of pPV-AB^2470-3386 and pT7PVM with one another produced pPV-AB^2954-3386 and pPV-AB^2470-2954, respectively.

Cloning of dicistronic reporter constructs was accomplished by first introducing a silent mutation in pHRPF-Luc by site-directed mutagenesis using oligonucleotides Fluc-mutRI(+)/Fluc-mutRI(−) to mutate an EcoRI site in the firefly luciferase open reading frame and generate pdiLuc-mRI. The capsid regions of pT7PVM, pPV-AB^1513-2470 and pPV-AB^2470-2954 were PCR amplified using oligonucleotides RI-2A-P1wt(+)/P1wt-2A-RI(−). Capsid sequences of pPV-AB^2470-3386 and pPV-AB^2954-3386 or pPV-AB were amplified with RI-2A-P1 wt(+)/P1AB-2A-RI(−) or RI-2A-P1 AB(+)/P1 AB-2A-RI(−), respectively. PCR products were digested with EcoRI and inserted into a now unique EcoRI site in pdiLuc-mRI to result in pdiLuc-PV, pdiLuc-AB^1513-2470, pdiLuc-AB^2470-2954, pdiLuc-AB^2470-3386, pdiLuc-AB^2954-3386, and pdiLuc-AB, respectively.

Oligonucleotides

The following oligonucleotides were used:

Fluc-mutRI(+),
(SEQ ID NO: 6)
5′-GCACTGATAATGAACTCCTCTGGATCTACTGG-3′;
Fluc-mutRI(−),
(SEQ ID NO: 7)
5′-CCAGTAGATCCAGAGGAGTTCATTATCAGTGC-3′;
RI-2A-P1wt(+),
(SEQ ID NO: 8)
5′-CAAGAATTCCTGACCACATACGGTGCTCAGGTTTCATCACAGAAA
GTGGG-3′;
RI-2A-P1AB(+),
(SEQ ID NO: 9)
5′-CAAGAATTCCTGACCACATACGGTGCGCAAGTATCGTCGCAAAAA
GTAGG-3;
P1wt-2A-RI(−),
(SEQ ID NO: 10)
5′-TTCGAATTCTCCATATGTGGTCAGATCCTTGGTGG-AGAGG-3′;
and
P1AB-2A-RI(−),
(SEQ ID NO: 11)
5′-TTCGAATTCTCCATACGTCGTTAAATCTTTCGTCGATAACG-3′.

In Vitro Transcription and RNA Transfection

Driven by the T7 promoter, 2 μg of EcoRI-linearized plasmid DNA were transcribed by T7 RNA polymerase (Stratagene) for 1 h at 37° C. One microgram of virus or dicistronic transcript RNA was used to transfect 10⁶ HeLa R19 cells on a 35-mm-diameter plate according to a modification of the DEAE-dextran method (van der Werf et al., 1986). Following a 30-min incubation at room temperature, the supernatant was removed and cells were incubated at 37° C. in 2 ml of DMEM containing 2% BCS until CPE appeared, or the cells were frozen 4 days post-transfection for further passaging. Virus titers were determined by standard plaque assay on HeLa R19 cells using a semisolid overlay of 0.6% tragacanth gum (Sigma-Aldrich) in minimal Eagle medium.

Design and Synthesis of Codon-Deoptimized Polioviruses

Two different synonymous encodings of the poliovirus P1 capsid region were produced, each governed by different design criteria. The designs were limited to the capsid, as it has been conclusively shown that the entire capsid coding sequence can be deleted from the PV genome or replaced with exogenous sequences without affecting replication of the resulting sub-genomic replicon (Johansen and Morrow, 2000; Kaplan and Racaniello, 1988). It is therefore quite certain that no unidentified crucial regulatory RNA elements are located in the capsid region, which might be affected inadvertently by modulation of the RNA sequence.

The first design (PV-SD) sought to maximize the number of RNA base changes while preserving the exact codon usage distribution of the wild type P1 region (FIG. 1). To achieve this, synonymous codon positions were exchanged for each amino acid by finding a maximum weight bipartite match (Gabow, 1973) between the positions and the codons, where the weight of each position-codon pair is the number of base changes between the original codon and the synonymous candidate codon to replace it. To avoid any positional bias from the matching algorithm, the synonymous codon locations were randomly permuted before creating the input graph and the locations were subsequently restored. Rothberg's maximum bipartite matching program (Rothberg, 1985) was used to compute the matching. A total of 11 useful restriction enzyme sites, each 6 nucleotides, were locked in the viral genome sequence so as to not participate in the codon location exchange. The codon shuffling technique potentially creates additional restriction sites that should preferably remain unique in the resulting reconstituted full-length genome. For this reason, the sequence was further processed by substituting codons to eliminate the undesired sites. This resulted in an additional nine synonymous codon changes that slightly altered the codon frequency distribution. However, no codon had its frequency changed by more than 1 over the wild type sequence. In total, there were 934 out of 2,643 nucleotides changed in the PV-SD capsid design when compared to the wt P1 sequence while maintaining the identical protein sequence of the capsid coding domain (see FIGS. 1 and 2). As the codon usage was not changed, the GC content in the PVM-SD capsid coding sequence remained identical to that in the wt at 49%.

The second design, PV-AB, sought to drastically change the codon usage distribution over the wt P1 region. This design was influenced by recent work suggesting that codon bias may impact tissue-specific expression (Plotkin et al., 2004). The desired codon usage distribution was derived from the most unfavorable codons observed in a previously described set of brain-specific genes (Hsiao et al., 2001; Plotkin et al., 2004). A capsid coding region was synthesized maximizing the usage of the rarest synonymous codon for each particular amino acid as observed in this set of genes (FIG. 1). Since for all amino acids but one (Leu) the rarest codon in brain corresponds to the rarest codons among all human genes at large, in effect this design would be expected to discriminate against expression in other human tissues as well. Altogether, the PV-AB capsid differs from the wt capsid in 680 nucleotide positions (see FIG. 2). The GC content in the PVM-AB capsid region was reduced to 43% compared to 49% in the wt.

Example 2

Effects of Codon-Deoptimization on Growth and Infectivity of Polioviruses

Determination of Virus Titer by Infected Focus Assay

Infections were done as for a standard plaque assay. After 48 or 72 h of incubation, the tragacanth gum overlay was removed and the wells were washed twice with phosphate-buffered saline (PBS) and fixed with cold methanol/acetone for 30 min Wells were blocked in PBS containing 10% BCS followed by incubation with a 1:20 dilution of anti-3D mouse monoclonal antibody 125.2.3 (Paul et al., 1998) for 1 h at 37° C. After washing, cells were incubated with horseradish peroxidase-labeled goat anti-mouse antibody (Jackson ImmunoResearch, West Grove, Pa.) and infected cells were visualized using Vector VIP substrate kit (Vector Laboratories, Burlingame, Calif.). Stained foci, which are equivalent to plaques obtained with wt virus, were counted, and titers were calculated as in the plaque assay procedure.

Codon-Deoptimized Polioviruses Display Severe Growth Phenotypes

Of the two initial capsid ORF replacement designs (FIG. 3A), only PV-SD produced viable virus. In contrast, no viable virus was recovered from four independent transfections with PV-AB RNA, even after three rounds of passaging (FIG. 3E). It appeared that the codon bias introduced into the PV-AB genome was too severe. Thus, smaller portions of the PV-AB capsid coding sequence were subcloned into the PV(M) background to reduce the detrimental effects of the nonpreferred codons. Of these subclones, PV-AB^2954-3386 produced CPE 40 h after RNA transfection, while PV-AB^755-1513 and PV-AB^2470-2954 required one or two additional passages following transfection, respectively (compared to 24 h for the wild type virus). Interestingly, these chimeric viruses represent the three subclones with the smallest portions of the original AB sequence, an observation suggesting a direct correlation between the number of nonpreferred codons and the fitness of the virus.

One-step growth kinetics of all viable virus variants were determined by infecting HeLa monolayers at a multiplicity of infection (MOI) of 2 with viral cell lysates obtained after a maximum of two passages following RNA transfection (FIG. 3B). The MOI was chosen due to the low titer of PV-AB^2470-2954 and to eliminate the need for further passaging required for concentrating and purifying the inoculum. Under the conditions used, all viruses had produced complete or near complete CPE by 24 h post-infection.

Despite 934 single-point mutations in its capsid region, PV-SD replicated at wt capacity (FIG. 3B) and produced similarly sized plaques as the wt (FIG. 3D). While PV-AB^2954-3386 grew with near-wild type kinetics (FIG. 3B), PV-AB^755-1513 produced minute plaques and approximately 22-fold less infectious virus (FIGS. 2. 3B and F, respectively). Although able to cause CPE in high-MOI infections, albeit much delayed (80 to 90% CPE after 20 to 24 h), PV-AB^2470-2954 produced no plaques at all under the conditions of the standard plaque assay (FIG. 3H). This virus was therefore quantified using a focus-forming assay, in which foci of infected cells after 72 h of incubation under plaque assay conditions were counted after they were stained immunohistochemically with antibodies to the viral polymerase 3D (FIG. 3G). After 48 h of infection, PV-AB^2470-2954-infected foci usually involved only tens to hundreds of cells (FIG. 3J) with a focus diameter of 0.2 to 0.5 mm, compared to 3-mm plaques for the wt (FIGS. 3C and D). However, after an additional 24 h, the diameter of the foci increased significantly (2 to 3 mm; FIG. 3G). When HeLa cells were infected with PV-AB^755-1513 and PV-AB^2470-2954 at an MOI of 1, the CPE appeared between 12 and 18 h and 3 and 4 days, respectively, compared to 8 h with the wt (data not shown).

In order to quantify the cumulative effect of a particular codon bias in a protein coding sequence, a relative codon deoptimization index (RCDI) was calculated, which is a comparative measure against the general codon distribution in the human genome. An RCDI of 1/codon indicates that a gene follows the normal human codon frequencies, while any deviation from the normal human codon bias results in an RCDI higher than 1. The RCDI was derived using the formula:RCDI=[Σ(C_iF_a/C_iF_h)·N_ci]/N (i=1 through 64).

C_iF_a is the observed relative frequency in the test sequence of each codon i out of all synonymous codons for the same amino acid (0 to 1); C_iF_h is the normal relative frequency observed in the human genome of each codon i out of all synonymous codons for that amino acid (0.06 to 1); N_ci is the number of occurrences of that codon i in the sequence; and N is the total number of codons (amino acids) in the sequence.

Thus, a high number of rare codons in a sequence results in a higher index. Using this formula, the RCDI values of the various capsid coding sequences were calculated to be 1.14 for PV(M) and PV-SD which is very close to a normal human distribution. The RCDI values for the AB constructs are 1.73 for PV-AB^755-1513, 1.45 for PV-AB^2470-2954, and 6.51 for the parental PV-AB. For comparison, the RCDI for probably the best known codon-optimized protein, “humanized” green fluorescent protein (GFP), was 1.31 compared to an RCDI of 1.68 for the original Aequora victoria gfp gene (Zolotukhin et al., 1996). According to these calculations, a capsid coding sequence with an RCDI of <2 is associated with a viable virus phenotype, while an RCDI of >2 (PV-AB=6.51, PV-AB^1513-3386=4.04, PV-AB^755-2470=3.61) results in a lethal phenotype.

Example 3

Effects of Codon-Deoptimization on Specific Infectivity of Polioviruses

Molecular Quantification of Viral Particles: Direct OD₂₆₀ Absorbance Method

Fifteen-centimeter dishes of HeLa cells (4×10⁷ cells) were infected with PV(M), PV-AB^755-1513, or PV-AB^2470-2954 at an MOI of 0.5 until complete CPE occurred (overnight versus 4 days). Cell-associated virus was released by three successive freeze/thaw cycles. Cell lysates were cleared by 10 min of centrifugation at 2,000×g followed by a second 10-min centrifugation at 14,000×g for 10 min Supernatants were incubated for 1 h at room temperature in the presence of 10 μg/ml RNase A (Roche) to digest any extraviral or cellular RNA. After addition of 0.5% sodium dodecyl sulfate (SDS) and 2 mM EDTA, virus-containing supernatants were overlaid on a 6-ml sucrose cushion (30% sucrose in Hanks balanced salt solution [HBSS]; Invitrogen, Carlsbad, Calif.). Virus particles were sedimented by ultracentrifugation for 4 h at 28,000 rpm using an SW28 swinging bucket rotor. Supernatants were discarded and centrifuge tubes were rinsed twice with HBSS while leaving the sucrose cushion intact. After removal of the last wash and the sucrose cushion, virus pellets were resuspended in PBS containing 0.2% SDS and 5 mM EDTA. Virus infectious titers were determined by plaque assay/infected-focus assay (see above). Virus particle concentrations were determined with a NanoDrop spectrophotometer (NanoDrop Technologies, Inc., Wilmington, Del.) at the optical density at 260 nm (OD₂₆₀) and calculated using the formula 1 OD₂₆₀ unit=9.4×10¹² particles/ml (Rueckert, 1985). In addition, virion RNA was extracted by three rounds of phenol extraction and one round of chloroform extraction. RNA was ethanol precipitated and resuspended in ultrapure water. RNA purity was confirmed by TAE-buffered agarose gel analysis, and the concentration was determined spectrophotometrically. The total number of genome equivalents of the corresponding virus preparation was calculated via the determined RNA concentration and the molecular weight of the RNA. Thus, the relative amount of virions per infectious units could be calculated, assuming that one RNase-protected viral genome equivalent corresponds to one virus particle.

Molecular Quantification of Viral Particles: ELISA Method

Nunc Maxisorb 96-well plates were coated with 10 μg of rabbit anti-PV(M) antibody (Murdin and Wimmer, 1989) in 100 μl PBS for 2 h at 37° C. and an additional 14 h at 4° C., and then the plates were washed three times briefly with 350 μl of PBS and blocked with 350 μl of 10% bovine calf serum in PBS for 1 h at 37° C. Following three brief washes with PBS, wells were incubated with 100 μl of virus-containing cell lysates or controls in DMEM plus 2% BCS for 4 h at room temperature. Wells were washed with 350 μl of PBS three times for 5 min each. Wells were then incubated for 4 h at room temperature with 2 μg of CD155-alkaline phosphatase (AP) fusion protein (He et al., 2000) in 100 μl of DMEM-10% BCS. After the last of five washes with PBS, 100 μl of 10 mM Tris, pH 7.5, were added and plates were incubated for 1 h at 65° C. Colorimetric alkaline phosphatase determination was accomplished by addition of 100 μl of 9 mg/ml para-nitrophenylphosphate (in 2 M diethanolamine, 1 mM MgCl₂, pH 9.8). Alkaline phosphatase activity was determined, and virus particle concentrations were calculated in an enzyme-linked immunosorbent assay (ELISA) plate reader (Molecular Devices, Sunnyvale, Calif.) at a 405-nm wavelength on a standard curve prepared in parallel using two-fold serial dilutions of a known concentration of purified PV(M) virus stock.

The PFU/Particle Ratio is Reduced in Codon-Deoptimized Viruses

The extremely poor growth phenotype of PV-AB^2470-2954 in cell culture and its inability to form plaques suggested a defect in cell-to-cell spreading that may be consistent with a lower specific infectivity of the individual virus particles.

To test this hypothesis, PV(M), PV-AB^755-1513, and PV-AB^2470-2954 virus were purified and the amount of virus particles was determined spectrophotometrically. Purified virus preparations were quantified directly by measuring the OD₂₆₀, and particle concentrations were calculated according to the formula 1 OD₂₆₀ unit=9.4×10¹² particles/ml (FIG. 4D) (Rueckert, 1985). Additionally, genomic RNA was extracted from those virions (FIG. 4A) and quantified at OD₂₆₀ (data not shown). The number of virions (1 virion=1 genome) was then determined via the molecular size of 2.53×10⁶ g/mol for genomic RNA. Specifically, virus was prepared from 4×10⁷ HeLa cells that were infected with 0.5 MOI of virus until the appearance of complete CPE, as described above. Both methods of particle determinations produced similar results (FIG. 4D). Indeed, it was found that PV(M) and PV-AB^755-1513 produced roughly equal amounts of virions, while PV-AB^2470-2954 produced between ⅓ (by the direct UV method (FIG. 4D) to ⅛ of the number of virions compared to PV(M) (by genomic RNA method [data not shown]). In contrast, the wt virus sample corresponded to approximately 30 times and 3,000 times more infectious units than PV-AB^755-1513 and PV-AB^2470-2954, respectively (FIG. 4D). In addition, capsid proteins of purified virions were resolved by SDS-polyacrylamide gel electrophoresis (PAGE) and visualized by silver staining (FIG. 4B). These data also support the conclusion that on a per-cell basis, PV-AB^2470-2954 and PV-AB^755-1513 produce similar or only slightly reduced amounts of progeny per cell (FIG. 4B, lane 3), while their PFU/particle ratio is reduced. The PFU/particle ratio for a virus can vary significantly depending on the methods to determine either plaques (cell type for plaque assay and the particular plaque assay technique) or particle count (spectrophotometry or electron microscopy). A PFU/particle ratio of 1/115 for PV1(M) was determined using the method described herein, which compares well to previous determinations of 1/272 (Joklik and Darnell, 1961) (done on HeLa cells) and 1/87 (Schwerdt and Fogh, 1957) (in primary monkey kidney cells).

Development of a Virion-Specific ELISA

To confirm the reduced PFU/particle ratio observed with codon-deoptimized polioviruses, a novel virion-specific ELISA was developed (FIGS. 4C and E) as a way to determine the physical amount of intact viral particles in a sample rather than the infectious titer, which is a biological variable. The assay is based on a previous observation that the ectodomain of the PV receptor CD155 fused to heat-stable placental alkaline phosphatase (CD155-AP) binds very tightly and specifically to the intact 160S particle (He et al., 2000). Considering that PV 130S particles (A particles) lose their ability to bind CD155 efficiently (Hogle, 2002), it is expected that no other capsid intermediate or capsid subunits would interact with CD155-AP, thus ensuring specificity for intact particles. In support of this notion, lysates from cells that were infected with a vaccinia virus strain expressing the P1 capsid precursor (Ansardi et al., 1993) resulted in no quantifiable signal (data not shown).

The ELISA method allows for the quantification of virus particles in a crude sample such as the cell lysate after infection, which should minimize possible alteration of the PFU/particle ratio by other mechanisms during sample handling and purification (thermal/chemical inactivation, oxidation, degradation, etc.). Under the current conditions, the sensitivity of this assay is approximately 10⁷ viral particles, as there is no signal amplification step involved. This, in turn, resulted in an exceptionally low background. With this ELISA, PV particle concentrations could be determined in samples by back calculation on a standard curve prepared with purified PV(M) of known concentration (FIG. 4E). The particle determinations by ELISA agreed well with results obtained by the direct UV method (FIG. 4D).

Implications of Results

The present study has demonstrated the utility of large-scale codon deoptimization of PV capsid coding sequences by de novo gene synthesis for the generation of attenuated viruses. The initial goal was to explore the potential of this technology as a tool for generating live attenuated virus vaccines. Codon-deoptimized viruses were found to have very low specific infectivity (FIG. 4). The low specific infectivity (that is the chance of a single virus particle to successfully initiate an infectious cycle in a cell) results in a more slowly spreading virus infection within the host. This in turn allows the host organism more time to mount an immune response and clear the infection, which is a most desirable feature in an attenuated virus vaccine. On the other hand, codon-deoptimized viruses generated similar amounts of progeny per cell as compared the wild type virus, while being 2 to 3 orders of magnitude less infectious (FIG. 4). This allows the production of virus particles antigenically indistinguishable from the wt as effectively and cost-efficiently as the production of the wt virus itself. However due to the low specific infectivity the actual handling and processing of such a virus preparation is much safer. Since, there are increasing concerns about the production of virulent virus in sufficient quantities under high containment conditions and the associated risk of virus escape from the production facility either by accident or by malicious intent. viruses as described herein may prove very useful as safer alternatives in the production of inactivated virus vaccines. Since they are 100% identical to the wt virus at the protein level, an identical immune response in hosts who received inactivated virus is guaranteed.

Example 4

Effects of Codon-Deoptimization on Neuropathogenicity of Polioviruses

Mouse Neuropathogenicity Tests

Groups of four to five CD155tg mice (strain Tg21) (Koike et al., 1991) between 6 and 8 weeks of age were injected intracerebrally with virus dilutions between 10² and 10⁶ PFU/focus-forming units (FFU) in 30 μl PBS. Fifty percent lethal dose (LD₅₀) values were calculated by the method of Reed and Muench (1938). Virus titers in spinal cord tissues at the time of death or paralysis were determined by plaque or infected-focus assay.

Codon-Deoptimized Polioviruses are Neuroattenuated on a Particle Basis in CD155tg Mice

To test the pathogenic potential of viruses constructed in this study, CD155 transgenic mice (Koike et al., 1991) were injected intracerebrally with PV(M), PV-SD, PV-AB^755-1513, and PV-AB^2470-2954 at doses between 10² and 10⁵ PFU/FFU. Initial results were perplexing, as quite counterintuitively PV-AB^755-1513 and especially PV-AB^2470-2954 were initially found to be as neuropathogenic as, or even slightly more neuropathogenic, than the wt virus. See Table 4.

TABLE 4
Neuropathogenicity in CD155tg mice.
	LD50	Spinal cord titer
Construct	PFU or FFUa	No. of virionsb	PFU or FFU/gc	No. of virions/gd
PV(M) wt	3.2 × 102 PFU	3.7 × 104	1.0 × 109 PFU	1.15 × 1011
PV-AB755-1515	2.6 × 102 PFU	7.3 × 105	3.5 × 107 PFU	9.8 × 1010
PV-AB2470-2954	4.6 × 102 PFU	4.8 × 106	3.4 × 106 FFU	3.57 × 1011
aLD50 expressed as the number of infectious units, as determined by plaque or infectious focus assay, that results in 50% lethality after intracerebral inoculation.
bLD50 expressed as the number of virus particles, as determined by OD260 measurement, that results in 50% lethality after intracerebral inoculation.
cVirus recovered from the spinal cord of infected mice at the time of death or paralysis; expressed in PFU or FFU/g of tissue, as determined by plaque or infectious focus assay.
dVirus recovered from the spinal cord of infected mice at the time of death or paralysis, expressed in particles/g of tissue, derived by multiplying values in the third column by the particle/PFU ratio characteristic for each virus (FIG. 4D).

In addition, times of onset of paralysis following infection with PV-AB^755-1513 and PV-AB^2470-2954 were comparable to that of wt virus (data not shown). Similarly confounding was the observation that at the time of death or paralysis, the viral loads, as determined by plaque assay, in the spinal cords of mice infected with PV-AB^755-1513 and PV-AB^2470-2954 were 30- and 300-fold lower, respectively, than those in the mice infected with the wt virus (Table 4). Thus, it seemed unlikely that PV-AB^2470-2954, apparently replicating at only 0.3% of the wt virus, would have the same neuropathogenic potential as the wt. However, after having established the altered PFU/particle relationship in PV-AB^755-1513 and PV-AB^2470-2954 (see Example 3), the amount of inoculum could now be correlated with the actual number of particles inoculated. After performing this correction, it was established that on a particle basis, PV-AB^755-1513 and PV-AB^2470-2954 are 20-fold and 100-fold neuroattenuated, respectively, compared to the wt. See Table 4. Furthermore, on a particle basis the viral loads in the spinal cords of paralyzed mice were very similar with all three viruses (Table 4).

It was also concluded that it was not possible to redesign the PV capsid gene with synonymous codons that would specifically discriminated against expression in the central nervous system. This may be because tissue-specific differences in codon bias described by others (Plotkin et al., 2004) are too small to bring about a tissue-restrictive virus phenotype. In a larger set of brain-specific genes than the one used by Plotkin et al., no appreciable tissue-specific codon bias was detected (data not shown). However, this conclusion should not detract from the fact that polioviruses produced by the method of this invention are indeed neuroattenuated in mice by a factor of up to 100 fold. That is, 100 fold more of the codon or codon-pair deoptimized viral particles are needed to result in the same damage in the central nervous system as the wt virus.

Example 5

Effects of Codon Deoptimization on Genomic Translation of Polioviruses

In Vitro and In Vivo Translation

Two different HeLa cell S10 cytoplasmic extracts were used in this study. A standard extract was prepared by the method of Molla et al. (1991). [³⁵S]methionine-labeled translation products were analyzed by gel autoradiography. The second extract was prepared as described previously (Kaplan and Racaniello, 1988), except that it was not dialyzed and endogenous cellular mRNAs were not removed with micrococcal nuclease. Reactions with the modified extract were not supplemented with exogenous amino acids or tRNAs. Translation products were analyzed by western blotting with anti-2C monoclonal antibody 91.23 (Pfister and Wimmer, 1999). Relative intensities of 2BC bands were determined by a pixel count of the scanned gel image using the NIH-Image 1.62 software. In all cases, translation reactions were programmed with 200 ng of the various in vitro-transcribed viral genomic RNAs.

For analysis of in vivo translation, HeLa cells were transfected with in vitro-transcribed dicistronic replicon RNA as described above. In order to assess translation isolated from RNA replication, transfections were carried out in the presence of 2 mM guanidine hydrochloride. Cells were lysed after 7 h in passive lysis buffer (Promega, Madison, Wis.) followed by a dual firefly (F-Luc) and Renilla (R-Luc) luciferase assay (Promega). Translation efficiency of the second cistron (P1-Fluc-P2-P3 polyprotein) was normalized through division by the Renilla luciferase activity of the first cistron expressed under control of the Hepatitis C Virus (HCV) internal ribosome entry site (IRES).

Codon-Deoptimized Viruses are Deficient at the Level of Genome Translation

Since the synthetic viruses and the wt PV(M) are indistinguishable in their protein makeup and no known RNA-based regulatory elements were altered in the modified RNA genomes, these designs enabled study of the effect of reduced genome translation/replication on attenuation without affecting cell and tissue tropism or immunological properties of the virus. The PV-AB genome was designed under the hypothesis that introduction of many suboptimal codons into the capsid coding sequence should lead to a reduction of genome translation. Since the P1 region is at the N-terminus of the polyprotein, synthesis of all downstream nonstructural proteins is determined by the rate of translation through the P1 region. To test whether in fact translation is affected, in vitro translations were performed (FIG. 5).

Unexpectedly, the initial translations in a standard HeLa-cell based cytoplasmic S10 extract (Molla et al., 1991) showed no difference in translation capacities for any of the genomes tested (FIG. 5A). However, as this translation system is optimized for maximal translation, it includes the exogenous addition of excess amino acids and tRNAs, which could conceivably compensate for the genetically engineered codon bias. Therefore, in vitro translations were repeated with a modified HeLa cell extract, which was not dialyzed and in which cellular mRNAs were not removed by micrococcal nuclease treatment (FIG. 5B). Translations in this extract were performed without the addition of exogenous tRNAs or amino acids. Thus, an environment was created that more closely resembles that in the infected cell, where translation of the PV genomes relies only on cellular supplies while competing for resources with cellular mRNAs. Due to the high background translation from cellular mRNA and the low [³⁵S]Met incorporation rate in nondialyzed extract, a set of virus-specific translation products were detected by western blotting with anti-2C antibodies (Pfister and Wimmer, 1999). These modified conditions resulted in dramatic reduction of translation efficiencies of the modified genomes which correlated with the extent of the deoptimized sequence. Whereas translation of PV-SD was comparable to that of the wt, translation of three noninfectious genomes, PV-AB, PV-AB^1513-3386, and PV-AB^755-2479, was reduced by approximately 90% (FIG. 5B).

Burns et al. (2006) recently reported experiments related to those described herein. These authors altered codon usage to a much more limited extent than in the present study, and none of their mutant viruses expressed a lethal phenotype. Interestingly, Burns et al. determined that translation did not play a major role in the altered phenotypes of their mutant viruses, a conclusion at variance with the data presented herein. It is likely that the in vitro translation assay used by Burns et al. (2006), which employed a nuclease-treated rabbit reticulocyte lysate supplemented with uninfected HeLa cell extract and excess amino acids, explains their failure to detect any significant reduction in translation. Cf. FIG. 5A.

Considering the ultimately artificial nature of the in vitro translation system, the effect of various capsid designs on translation in cells was also investigated. For this purpose, dicistronic poliovirus reporter replicons were constructed (FIG. 6A) based on a previously reported dicistronic replicon (Zhao and Wimmer, 2001). Various P1 cassettes were inserted immediately upstream and in-frame with the firefly luciferase (F-Luc) gene. Thus, the poliovirus IRES drives expression of a single viral polyprotein similar to the one in the viral genome, with the exception of the firefly luciferase protein between the capsid and the 2A^pro proteinase. Expression of the Renilla luciferase (R-Luc) gene under the control of the HCV IRES provides an internal control. All experiments were carried out in the presence of 2 mM guanidine hydrochloride, which completely blocks genome replication (Wimmer et al., 1993). Using this type of construct allowed an accurate determination of the relative expression of the second cistron by calculating the F-Luc/R-Luc ratio. As F-Luc expression depends on successful transit of the ribosome through the upstream P1 region, it provides a measure of the effect of the inserted P1 sequence on the rate of polyprotein translation. Using this method, it was indeed found that the modified capsid coding regions, which were associated with a lethal phenotype in the virus background (e.g., PV-AB, PV-AB^1513-2470, and PV-AB^2470-3386) reduced the rate of translation by approximately 80 to 90% (FIG. 6B). Capsids from two viable virus constructs, PV-AB^2470-2954 and PV-AB^2954-3386, allowed translation at 68% and 83% of wt levels, respectively. In vivo translation rates of the first cistron remained constant in all constructs over a time period between 3 and 12 h, suggesting that RNA stability is not affected by the codon alterations (data not shown). In conclusion, the results of these experiments suggest that poliovirus is extremely dependent on very efficient translation as a relatively small drop in translation efficiency through the P1 region of 30%, as seen in PV-AB^2470-2954, resulted in a severe virus replication phenotype.

Example 6

Genetic Stability of Codon-Deoptimized Polioviruses

Due to the distributed effect of many mutations over large genome segments that contribute to the phenotype, codon deoptimized viruses should have genetically stable phenotypes. To study the genetic stability of codon deoptimized viruses, and to test the premise that these viruses are genetically stable, viruses are passaged in suitable host cells. A benefit of the present “death by 1000 cuts” theory of vaccine design is the reduced risk of reversion to wild type. Typical vaccine strains differ by only few point mutations from the wt viruses, and only a small subset of these may actually contribute to attenuation. Viral evolution quickly works to revert such a small number of active mutations. Indeed, such reversion poses a serious threat for the World Health Organization (WHO) project to eradicate poliovirus from the globe. So long as a live vaccine strain is used, there is a very real chance that this strain will revert to wt. Such reversion has already been observed as the source of new polio outbreaks (Georgescu et al., 1997; Kew et al., 2002; Shimizu et al., 2004).

With hundreds to thousands of point mutations in the present synthetic designs, there is little risk of reversion to wt strains. However, natural selection is powerful, and upon passaging, the synthetic viruses inevitably evolve. Studies are ongoing to determine the end-point of this evolution, but a likely outcome is that they get trapped in a local optimum, not far from the original design.

To validate this theory, representative re-engineered viruses are passaged in a host cell up to 50 times. The genomes of evolved viruses are sequenced after 10, 20 and 50 passages. More specifically, at least one example chimera from each type of deoptimized virus is chosen. The starting chimera is very debilitated, but not dead. For example, for PV the chimeras could be PV-AB^2470-2954 and PV-Min^755-2470. From each starting virus ten plaques are chosen. Each of the ten plaque-derived virus populations are bulk passaged a total of 50 times. After the 10^th, 20^th and 50^th passages, ten plaque-purified viruses are again chosen and their genomes are sequenced together with the genomes of the ten parent viruses. After passaging, the fitness of the 40 (30+10 per parent virus) chosen viruses is compared to that of their parents by examining plaque size, and determining plaque forming units/ml as one-step growth kinetics. Select passage isolates are tested for their pathogenicity in appropriate host organisms. For example, the pathogenicity of polioviruses is tested in CD155tg mice.

Upon sequencing of the genomes, a finding that all 10 viral lines have certain mutations in common would suggest that these changes are particularly important for viral fitness. These changes may be compared to the sites identified by toeprinting as the major pause sites (see Example 9); the combination of both kinds of assay may identify mutant codons that are most detrimental to viral fitness. Conversely, a finding that the different lines have all different mutations would support the view that many of the mutant codon changes are very similar in their effect on fitness. Thus far, after 10 passages in HeLa cells, PV-AB^755-1513 and PV-AB^2470-2954 have not undergone any perceivable gain of fitness. Viral infectious titers remained as low (10⁷ PFU/ml and 10⁶ FFU/ml) as at the beginning of the passage experiment, and plaque phenotype did not change (data not shown). Sequence analysis of these passaged viruses is now in progress, to determine if and what kind of genetic changes occur during passaging.

Burns et al. (2006) reported that their altered codon compositions were largely conserved during 25 serial passages in HeLa cells. They found that whereas the fitness for replication in HeLa cells of both the unmodified Sabin 2 virus and the codon replacement viruses increased with higher passage numbers, the relative fitness of the modified viruses remained lower than that of the unmodified virus. Thus, all indications are that viruses redesigned by SAVE are genetically very stable. Preliminary data for codon and codon-pair deoptimized viruses of the invention suggest that less severe codon changes distributed over a larger number of codons improves the genetic stability of the individual virus phenotypes and thus improves their potential for use in vaccines.

Example 7

Re-Engineering of Capsid Region of Polioviruses by Deoptimizing Codon Pairs

Calculation of Codon Pair Bias.

Every individual codon pair of the possible 3721 non-“STOP” containing codon pairs (e.g., GTT-GCT) carries an assigned “codon pair score,” or “CPS” that is specific for a given “training set” of genes. The CPS of a given codon pair is defined as the log ratio of the observed number of occurrences over the number that would have been expected in this set of genes (in this example the human genome). Determining the actual number of occurrences of a particular codon pair (or in other words the likelihood of a particular amino acid pair being encoded by a particular codon pair) is simply a matter of counting the actual number of occurrences of a codon pair in a particular set of coding sequences. Determining the expected number, however, requires additional calculations. The expected number is calculated so as to be independent of both amino acid frequency and codon bias similarly to Gutman and Hatfield. That is, the expected frequency is calculated based on the relative proportion of the number of times an amino acid is encoded by a specific codon. A positive CPS value signifies that the given codon pair is statistically over-represented, and a negative CPS indicates the pair is statistically under-represented in the human genome.

To perform these calculations within the human context, the most recent Consensus CDS (CCDS) database of consistently annotated human coding regions, containing a total of 14,795 genes, was used. This data set provided codon and codon pair, and thus amino acid and amino-acid pair frequencies on a genomic scale.

The paradigm of Federov et al. (2002), was used to further enhanced the approach of Gutman and Hatfield (1989). This allowed calculation of the expected frequency of a given codon pair independent of codon frequency and non-random associations of neighboring codons encoding a particular amino acid pair.

$S\left(P_{\mathrm{ij}}\right)=\ln \left(\frac{N_O\left(P_{\mathrm{ij}}\right)}{N_E\left(P_{\mathrm{ij}}\right)}\right)=\ln \left(\frac{N_O\left(P_{\mathrm{ij}}\right)}{F\left(C_i\right)F\left(C_j\right)N_O\left(X_{\mathrm{ij}}\right)}\right)$

In the calculation, P_ij is a codon pair occurring with a frequency of N_O(P_ij) in its synonymous group. C_i and C_j are the two codons comprising P_ij, occurring with frequencies F(C_i) and F(C_j) in their synonymous groups respectively. More explicitly, F(C_i) is the frequency that corresponding amino acid X_i is coded by codon C_i throughout all coding regions and F(C_i)=N_O(C_i)/N_O(X_i), where N_O(C_i) and N_O(X_j) are the observed number of occurrences of codon C_i and amino acid X_i respectively. F(C_j) is calculated accordingly. Further, N_O(X_ij) is the number of occurrences of amino acid pair X_ij throughout all coding regions. The codon pair bias score S(P_ij) of P_ij was calculated as the log-odds ratio of the observed frequency N_O(P_ij) over the expected number of occurrences of N_e(P_ij).

Using the formula above, it was then determined whether individual codon pairs in individual coding sequences are over- or under-represented when compared to the corresponding genomic N_e(P_ij) values that were calculated by using the entire human CCDS data set. This calculation resulted in positive S(P_ij) score values for over-represented and negative values for under-represented codon pairs in the human coding regions (FIG. 7).

The “combined” codon pair bias of an individual coding sequence was calculated by averaging all codon pair scores according to the following formula:

$S\left(P_{\mathrm{ij}}\right)=\sum _{l=1}^k\frac{S\left(\mathrm{Pij}\right)l}{k-1}.$

The codon pair bias of an entire coding region is thus calculated by adding all of the individual codon pair scores comprising the region and dividing this sum by the length of the coding sequence.

Changing of Codon Pair Bias.

The capsid-coding region of PV(M) was re-engineered to change codon pair bias. The largest possible number of rarely used codon pairs (creating virus PV-Min) or the largest possible number of widely used codon pairs (creating virus PV-Max) was introduced, while preserving the codon bias and all other features of the wt virus genome. The following explains our method in detail.

Two sequences were designed to vary the poliovirus P1 region codon pair score in the positive (PV-Max; SEQ ID NO:4) and negative (PV-Min; SEQ ID NO:5) directions. By leaving the amino acid sequence unaltered and the codon bias minimally modified, a simulated annealing algorithm was used for shuffling codons, with the optimization goal of a minimum or maximum codon pair score for the P1 capsid region. The resulting sequences were processed for elimination of splice sites and reduction of localized secondary structures. These sequences were then synthesized by a commercial vendor, Blue Heron Biotechnology, and sequence-verified. The new capsid genes were used to replace the equivalent wt sequence in an infectious cDNA clone of wt PV via two PflMI restriction sites. Virus was derived as described in Example 1.

For the PV-Max virus, death of infected cells was seen after 24 h, a result similar to that obtained with wt virus. Maximal viral titer and one-step growth kinetics of PV-Max were also identical to the wt. In contrast, no cell death resulted in cells transfected with PV-Min mutant RNA and no viable virus could be recovered. The transfections were repeated multiple times with the same result. Lysates of PV-Min transfected cells were subjected to four successive blind passages, and still no virus was obtained.

The capsid region of PV-Min was divided into two smaller sub-fragments (PV-Min^755-2470 and PV-Min^2470-3386) as had been done for PV-AB (poor codon bias), and the sub-fragments were cloned into the wt background. As with the PV-AB subclones, subclones of PV-Min were very sick, but not dead (FIG. 8). As observed with PV-AB viruses, the phenotype of PV-Min viruses is a result of reduced specific infectivity of the viral particles rather than to lower production of progeny virus. Ongoing studies involve testing the codon pair-attenuated chimeras in CD155tg mice to determine their pathogenicity. Also, additional chimeric viruses comprising subclones of PV-Min cDNAs are being made, and their ability to replicate is being determined (see example 8 and 9 below). Also, the effect of distributing intermediate amounts of codon pair bias over a longer sequence are being confirmed. For example, a poliovirus derivative is designed to have a codon pair bias of about −0.2 (PV-0.2; SEQ ID NO:6), and the mutations from wild type are distributed over the full length of the P1 capsid region. This is in contrast to PV-MinZ (PV-Min^2470-3386) which has a similar codon pair bias, but with codon changes distributed over a shorter sequence.

It is worth pointing out that PV-Min and PV-0.2 are sequences in which there is little change in codon usage relative to wild type. For the most part, the sequences employ the same codons that appear in the wild type PV(M) virus. PV-MinZ is somewhat different in that it contains a portion of PV-Min subcloned into PV(M). As with PV-Min and PV-0.2, the encoded protein sequence is unchanged, but codon usage as determined in either the subcloned region, or over the entire P1 capsid region, is not identical to PV-Min (or PV-0.2), because only a portion of the codon rearranged sequence (which has identical codons over its full length, but not within smaller segments) has been substituted into the PV(M) wild type sequence. Of course, a mutated capsid sequence could be designed to have a codon pair bias over the entire P1 gene while shuffling codons only in the region from nucleotides 2470-3386.

Example 8

Viruses Constructed by a Change of Codon-Pair Bias are Attenuated in CD155 tg Mice

Mice Intracerebral Injections, Survival

To test the attenuation of PV-Min^755-2470 and PV-Min^2470-3385 in an animal model, these viruses were purified and injected intra-cerebrally into CD 155 (PVR/poliovirus receptor) transgenic mice (See Table 5). Indeed these viruses showed a significantly attenuated phenotype due to the customization of codon pair bias using our algorithm PVM-wt was not injected at higher dose because all mice challenged at 10e5 virions died because of PVM-wt. This attenuated phenotype is due to the customization of codon pair bias using our algorithm. This reaffirms that the customization of codon-pair bias is applicable for a means to create live vaccines.

TABLE 5
Mice Intracerebral Injections, Survival.
	10e4
Virus	Virions	10e5 Virions	10e6 Virions	10e7 Virions
PV-Min755-2470	4/4	3/4	3/5	3/4
PV-Min2470-3385	4/4	4/4	5/5	3/4
PVM-wt	3/4	0/4	—	—

These findings are significant in two respects. First, they are the first clear experimental evidence that codon pair bias is functionally important, i.e., that a deleterious phenotype can be generated by disturbing codon pair bias. Second, they provide an additional dimension of synonymous codon changes that can be used to attenuate a virus. The in vivo pathogenicity of these codon-pair attenuated chimeras have been tested in CD155tg and have shown an attenuated phenotype (See Table 5). Additional chimeric viruses comprising subclones of PV-Min capsid cDNAs have been assayed for replication in infected cells and have also shown an attenuated phenotype.

Example 9

Construction of Synthetic Poliovirus with Altered Codon-Pair Bias: Implications for Vaccine Development

Calculation of Codon Pair Bias, Implementation of Algorithm to Produce Codon Pair Deoptimized Sequences.

We developed an algorithm to quantify codon pair bias. Every possible individual codon pair was given a “codon pair score”, or “CPS”. We define the CPS as the natural log of the ratio of the observed over the expected number of occurrences of each codon pair over all human coding regions.

$\mathrm{CPS}=\ln \left(\frac{{F\left(\mathrm{AB}\right)}_O}{\frac{F\left(A\right)\times F\left(B\right)}{F\left(X\right)\times F\left(Y\right)}\times F\left(\mathrm{XY}\right)}\right)$ Although the calculation of the observed occurrences of a particular codon pair is straightforward (the actual count within the gene set), the expected number of occurrences of a codon pair requires additional calculation. We calculate This expected number is calculated to be independent both of amino acid frequency and of codon bias, similar to Gutman and Hatfield. That is, the expected frequency is calculated based on the relative proportion of the number of times an amino acid is encoded by a specific codon. A positive CPS value signifies that the given codon pair is statistically over-represented, and a negative CPS indicates the pair is statistically under-represented in the human genome

Using these calculated CPSs, any coding region can then be rated as using over- or under-represented codon pairs by taking the average of the codon pair scores, thus giving a Codon Pair Bias (CPB) for the entire gene.

$\mathrm{CPB}=\sum _{i=1}^k\frac{\mathrm{CPSi}}{k-1}$ The CPB has been calculated for all annotated human genes using the equations shown and plotted (FIG. 7). Each point in the graph corresponds to the CPB of a single human gene. The peak of the distribution has a positive codon pair bias of 0.07, which is the mean score for all annotated human genes. Also there are very few genes with a negative codon pair bias. Equations established to define and calculate CPB were then used to manipulate this bias.

Development and Implementation of Computer-Based Algorithm to Produce Codon Pair Deoptimized Sequences.

Using these formulas we next developed a computer based algorithm to manipulate the CPB of any coding region while maintaining the original amino acid sequence. The algorithm has the critical ability to maintain the codon usage of a gene (i.e. preserve the frequency of use of each existing codon) but “shuffle” the existing codons so that the CPB can be increased or decreased. The algorithm uses simulated annealing, a mathematical process suitable for full-length optimization (Park, S. et al., 2004). Other parameters are also under the control of this algorithm; for instance, the free energy of the folding of the RNA. This free energy is maintained within a narrow range, to prevent large changes in secondary structure as a consequence of codon re-arrangement. The optimization process specifically excludes the creation of any regions with large secondary structures, such as hairpins or stem loops, which could otherwise arise in the customized RNA. Using this computer software the user simply needs to input the cDNA sequence of a given gene and the CPB of the gene can be customized as the experimenter sees fit.

De Novo Synthesis of P1 Encoded by Either Over-Represented or Under-Represented Codon-Pairs.

To obtain novel, synthetic poliovirus with its P1 encoded by either over-represented or under-represented codon pairs, we entered the DNA sequence corresponding to the P1 structural region of poliovirus type I Mahoney (PV(M)-wt) into our program yielding—PV-Max-P1 using over-represented codon pairs (566 mutations) and PV-Min-P1 using under-represented codon pairs (631 mutations). The CPB scores of these customized, novel synthetic P-1 regions are PV-Max=+0.25 and PV-Min=−0.48, whereas the CPB of PV(M)-wt is −0.02 (FIG. 7).

Additional customization included inclusion of restriction sites that were designed into both synthetic sequences at given intervals, to allow for sub-cloning of the P1 region. These synthetic P1 fragments were synthesized de novo by Blue Herron Corp. and incorporated into a full-length cDNA construct of poliovirus (FIG. 11) (Karlin et al., 1994). A small fragment (3 codons, 9 nucleotides) of PV(M)-wt sequence was left after the AUG start codon in both constructs to allow translation to initiate equally for all synthetic viruses; thus providing more accurate measurement of the effect of CPB on the elongation phase of translation.

DNA Synthesis, Plasmids, Sub Cloning of Synthetic Capsids and Bacteria.

Large codon-pair altered PV cDNA fragments, corresponding to nucleotides 495 to 3636 of the PV genome, were synthesized by Blue Heron Corp. using their proprietary GeneMaker® system (http://www.blueheronbio.com/). All subsequent poliovirus cDNA clones/sub clones were constructed from PV1(M) cDNA clone pT7PVM using unique restriction sites (van der Wert, et al., 1986). The full-length PV-Min, PV-Max cassette was released from Blue Heron's carrier vector via PflMI digestion and insertion into the pT7PVM vector with its PflMI fragment removed. The PV-MinXY and PV-MinZ constructs were obtained by digestion with NheI and BglII simultaneously, then swapping this fragment with a pT7PVM vector digested similarly PV-MinXY and PV-MinZ were constructed via BsmI digestion and exchanging the fragment/vector with the similarly digested pT7PVM. PV-MinY was constructed by digesting the PV-MinXY construct with BsmI and swapping this fragment with the BsmI fragment for a digested pT7PVM. Plasmid transformation and amplification were all achieved via Escherichia coli DH5α.

Creation of Chimeric Viruses Containing CPB-Altered Capsid Regions: Under-Represented Codon Pair Bias Throughout the P1 Results in a Null Phenotype.

Using the T7 RNA polymerase promoter upstream of the poliovirus genomic sequence, positive-sense RNA was transcribed. 1.5 μg of a given plasmid cDNA clone from above was linearized via an EcoRI digestion and than was transcribed into RNA via T7 RNA polymerase (Stratagene) driven by its promoter upstream of the cDNA for 2 hours at 37° C. (van der Werf et al., 1986). This RNA was transfected into 1×10⁶ HeLa R19 cells using a modified DEAE-Dextran method (van der Werf et al., 1986). These cells were than incubate at room-temperature (RT) for 30-minutes. The transfection supernatant was removed and Dulbecco's modified Eagle medium (DMEM) containing 2% bovine calf serum (BCS) was added and the cells were incubated at 37° C. and observed (up to 4 days) for the onset of cytopathic effect (CPE).

The PV-Max RNA transfection produced 90% cytopathic effect (CPE) in 24 hours, which is comparable to the transfection of PV(M)-wt RNA. The PV-Max virus generated plaques identical in size to the wild type. In contrast, the PV-Min RNA produced no visible cytopathic effect after 96 hours, and no viable virus could be isolated even after four blind passages of the supernatant from transfected cells.

The subsequent use of the supernatant from cells subjected to PV-Max RNA transfection also produced 95% CPE in 12 hours, thus indicating that the transfected genomic material successfully produced PV-Max poliovirus virions. In contrast, the PV-Min viral RNA yielded no visible CPE after 96 hours and four blind passages of the supernatant, possibly containing extremely low levels of virus, also did not produce CPE. Therefore the full-length PV-Min synthetic sequence, utilizing under-represented codon pairs, in the P1 region cannot generate viable virus and so it would need to be sub-cloned.

HeLa R19 cells were maintained as a monolayer in DMEM containing 10% BCS. Virus amplification was achieved on (1.0×10⁸ cells) HeLa R19 monolayers using 1 M.O.I. Infected cells were incubated at 37° C. in DMEM with 2% BCS for three days or until CPE was observed. After three freeze/thaw cycles cell debris was removed form the lysates via low speed centrifugation and the supernatant containing virus was used for further experiments.

One-Step growth curves were achieved by infecting a monolayer of HeLa R19 cells with 5 M.O.I of a given virus, the inoculums was removed, cells washed 2× with PBS and then incubating at 37° C. for 0, 2, 4, 7, 10, 24, and 48 hours. These time points were then analyzed via plaque assay. All Plaque assay were performed on monolayers of HeLa R19 cells. These cells were infected with serial dilution of a given growth curve time point or purified virus. These cells were then overlaid with a 0.6% tragenthum gum in Modified Eagle Medium containing 2% BCS and then incubated at 37° C. for either 2 days for PV(M)-wt and PV-Max, or 3 days for PV-Min (X, Y, XY, or Z) viruses. These were then developed via crystal violet staining and the PFU/ml titer was calculated by counting visible plaques.

Small Regions of Under-Represented Codon Pair Bias Rescues Viability, but Attenuate the Virus.

Using the restriction sites designed within the PV-Min sequence we subcloned portions of the PV-Min P1 region into an otherwise wild-type virus, producing chimeric viruses where only sub-regions of P1 had poor codon pair bias (FIG. 11) (van der Werf et al., 1986). From each of these sub-clones, RNA was produced via in vitro transcription and then transfected into HeLa R19 cells, yielding viruses with varying degrees of attenuation (Viability scores, FIG. 11). P1 fragments X and Y are each slightly attenuated; however when added together they yield a virus (PV-Min^755-2470, PV-MinXY) that is substantially attenuated (FIGS. 3, 4). Virus PVMin^2470-3385 (PV-MinZ) is about as attenuated as PV-MinXY. Construct PV-Min^1513-3385 (YZ) did not yield plaques, and so apparently is too attenuated to yield viable virus. These virus constructs, which displayed varying degrees of attenuation were further investigated to determine their actual growth kinetics.

One-Step Growth Kinetics and the Mechanism of Attenuation: Specific Infectivity is Reduced.

For each viable construct, one step-growth kinetics were examined. These kinetics are generally similar to that of wild-type in that they proceed in the same basic manner (i.e. an eclipse phase followed by rapid, logarithmic growth). However, for all PV-Min constructs, the final titer in terms of Plaque Forming Units (PFU) was typically lower than that of wild-type viruses by one to three orders of magnitude (FIG. 12A).

When virus is measured in viral particles per ml (FIG. 12B) instead of PFU, a slightly different result is obtained and suggests these viruses produce nearly equivalent numbers of particles per cell per cycle of infection as the wild-type virus. In terms of viral particles per ml, the most attenuated viruses are only 78% (PV-MinXY) or 82% (PV-MinZ) attenuated which on a log scale is less than one order of magnitude. Thus these viruses appear to be attenuated by about two orders of magnitude in their specific infectivity (the number of virions required to generate a plaque).

To confirm that specific infectivity was reduced, we re-measured the ratio of viral particles per PFU using highly purified virus particles. Selected viruses were amplified on 10⁸ HeLa R19 cells. Viral lysates were treated with RNAse A to destroy exposed viral genomes and any cellular RNAs, that would obscure OD values. Also the viral lysates were then incubated for 1 hour with 0.2% SDS and 2 min EDTA to denature cellular and non-virion viral proteins. A properly folded and formed poliovirus capsid survives this harsh SDS treatment, were as alph particles do not (Mueller et al., 2005). Virions from these treated lysates were then purified via ultracentrifugation over a sucrose gradient. The virus particle concentration was measured by optical density at 260 nm using the formula 9.4×10¹² particles/ml=1 OD₂₆₀ unit (Rueckert, 1985). A similar number of particles was produced for each of the four viruses (Table 6). A plaque assay was then performed using these purified virions. Again, PV-MinXY and PV-MinZ required many more viral particles than wild-type to generate a plaque (Table 6).

For wild-type virus, the specific infectivity was calculated to be 1 PFU per 137 particles (Table 6), consistent with the literature (Mueller et al., 2006; Schwerdt and Fogh, 1957; Joklik and Darnell, 1961). The specific infectivities of viruses PV-MinXY and PV-MinZ are in the vicinity of 1 PFU per 10,000 particles (Table 6).

Additionally the heat stability of the synthetic viruses was compared to that of PV(M)-wt to reaffirm the SDS treatment data, that these particles with portions of novel RNA were equally as stable. Indeed these synthetic viruses had the same temperature profile as PV(M)-wt when incubated at 50° C. and quantified as a time course (data not shown).

Under-Represented Codon Pairs Reduce Translation Efficiency, Whereas Over-Represented Pairs Enhance Translation.

One hypothesis for the existence of codon pair bias is that the utilization of under-represented pairs causes poor or slow translation rates. Our synthetic viruses are, to our knowledge, the first molecules containing a high concentration of under-represented codon pairs, and as such are the first molecules suitable for a test of the translation hypothesis.

To measure the effect of codon pair bias on translation, we used a dicistronic reporter (Mueller et al., 2006) (FIG. 13). The first cistron expresses Renilla luciferase (R-Luc) under the control of the hepatitis C virus internal ribosome entry site (IRES) and is used as a normalization control. The second cistron expresses firefly luciferase (F-Luc) under the control of the poliovirus IRES. However, in this second cistron, the F-Luc is preceded by the P1 region of poliovirus, and this P1 region could be encoded by any of the synthetic sequence variants described here. Because F-Luc is translated as a fusion protein with the proteins of the P1 region, the translatability of the P1 region directly affects the amount of F-Luc protein produced. Thus the ratio of F-Luc luminescence to R-Luc luminescence is a measure of the translatability of the various P1 encodings.

The P1 regions of wild-type, PV-Max, PV-Min, PV-MinXY and PV-MinZ were inserted into the region labeled “P1” (FIG. 13A). PV-MinXY, PV-MinZ, and PV-Min produce much less F-Luc per unit of R-Luc than does the wild-type P1 region, strongly suggesting that the under-represented codon pairs are causing poor or slow translation rates (FIG. 13). In contrast, PV-Max P1 (which uses over-represented codon pairs) produced more F-Luc per unit of R-Luc, suggesting translation is actually better for PV-Max P1 compared to PV(M)-wt P1.

Dicistronic Reporter Construction, and In Vivo Translation.

The dicistronic reporter constructs were all constructed based upon pdiLuc-PV (Mueller et al., 2006). PV-Max and PV-Min capsid regions were amplified via PCR using the oligonucleotides P1max-2A-RI (+)/P1max-2A-RI (−) or P1min-2A-RI (+)/P1min-2A-RI (−) respectively. The PCR fragment was gel purified and then inserted into an intermediate vector pCR-®-XL-TOPO® (Invitrogen). This intermediate vector was than amplified in One Shot® TOP10 chemically competent cells. After preparation of the plasmid via Quiagne miniprep the intermediate vectors containing PV-Min was digested with EcoRI and these fragments were ligated into the pdiLuc-PV vector that was equally digested with EcoRI (Mueller et al., 2006). These plasmids were also amplified in One Shot® TOP10 chemically competent cells (Invitrogen). To construct pdiLuc-PV-MinXY and pdiLuc-PV-MinZ, pdiLuc-PV and pdiLuc-PV-Min were equally digested with NheI and the resulting restriction fragments were exchanged between the respective vectors. These were than transformed into One Shot® TOP10 chemically competent cells and then amplified. From all four of these clones RNA was transcribed via the T7 polymerase method (van der Werf et al., 1986).

To analyze the in vivo translation efficiency of the synthetic capsids the RNA of the dicistronic reporter constructs were transfected into 2×10⁵ HeLa R19 cells on 12-well dishes via Lipofectamine 2000 (Invitrogen). In order to quantify the translation of only input RNA the transfection was accomplished in the presence of 2 min guanidine hydrochloride (GuHCL). Six hours after transfection cells were lysed via passive lysis buffer (Promega) and then these lysates were analyzed by a dual firefly (F-Luc) Renilla (R-Luc) luciferase assay (Promega).

Genetic Stability of PV-MinXY and PV-MinZ.

Because PV-MinXY and PV-MinZ each contain hundreds of mutations (407 and 224, respectively), with each mutation causing a miniscule decrease in overall codon pair bias, we believe it should be very difficult for these viruses to revert to wild-type virulence. As a direct test of this idea, viruses PV-MinXY and PV-MinZ were serially-passaged 15 times, respectively, at an MOI of 0.5. The titer was monitored for phenotypic reversion, and the sequence of the passaged virus was monitored for reversions or mutation. After 15 passages there was no phenotypic change in the viruses (i.e. same titer, induction of CPE) and there were no fixed mutations in the synthetic region.

Heat Stability and Passaging.

The stability of the synthetic viruses, PV-MinXY and PV-Min Z, was tested and compared to PV(M)-wt. This was achieved by heating 1×10⁸ particles suspended in PBS to 50° C. for 60 minutes and then measuring the decrease in intact viral particles via plaque assay at 5, 15, 30 and 60 minutes (FIG. 14). In order to test the genetic stability of the synthetic portions of the P1 region of the viruses PV-MinXY and PV-MinZ these viruses were serial passaged. This was achieved by infecting a monolayer of 1×10⁶ HeLa R19 cells with 0.5 MOI of viruses, PV-MinXY and PV-MinZ, and then waiting for the induction of CPE. Once CPE initiated, which remained constant throughout passages, the lysates were used to infect new monolayers of HeLa R19 cells. The titer and sequence was monitored at passages 5, 9, and 15 (data not shown).

Virus Purification and Determination of Viral Particles Via OD₂₆₀ Absorbance.

A monolayer of HeLa R19 cells on a 15 cm dish (1×10⁸ cells) were infected with PV(M)-wt, PV-Max, PV-MinXY or PV-Min Z until CPE was observed. After three freeze/thaw cycles the cell lysates were subjected to two initial centrifugations at 3,000×g for 15 minutes and then 10,000×g for 15 minutes. Then 10 μg/ml of RNAse A (Roche) was added to supernatant and incubated at RT for 1 hour; Subsequently 0.5% sodium dodecyl sulfate (SDS) and 2 mM EDTA was added to the supernatant, gently mixed and incubated at RT for 30 minutes. These supernatants containing virus particles were placed above a 6 ml sucrose cushion [30% sucrose in Hank's Buffered Salt Solution (HBSS)]. Sedimentation of virus particles was achieved by ultracentrifugation through the sucrose gradient for 3.5 hours at 28,000 rpm using an SW28 swing-bucket rotor.

After centrifugation, the sucrose cushion was left intact and the supernatant was removed and the tube was washed two times with HBBS. After washing, the sucrose was removed and the virus “pearl” was re-suspended in PBS containing 0.1% SDS. Viral titers were determined via plaque assay (above). Virus particles concentration was determined via the average of three measurements of the optical density at 260 nm of the solution via the NanoDrop spectrophotometer (NanoDrop Technologies) using the formula 9.4×10¹² particles/ml=1 OD₂₆₀ unit (Mueller et al., 2006; Rueckert, 1985).

Neuroattenuation of PV-MinXY and PV-MinZ in CD155tg Mice.

The primary site of infection of wild-type poliovirus is the oropharynx and gut, but this infection is relatively asymptomatic. However, when the infection spreads to motor neurons in the CNS in 1% of PV(M)-wt infections, the virus destroys these neurons, causing death or acute flaccid paralysis know as poliomyelitis (Landsteiner and Popper, 1909; Mueller et al., 2005). Since motor neurons and the CNS are the critical targets of poliovirus, we wished to know whether the synthetic viruses were attenuated in these tissues. Therefore these viruses were administered to CD155tg mice (transgenic mice expressing the poliovirus receptor) via intracerebral injection (Koike et al., 1991). The PLD₅₀ value was calculated for the respective viruses and the PV-MinXY and PV-MinZ viruses were attenuated either 1,000 fold based on particles or 10 fold based on PFU (Table 6) (Reed and Muench, 1938). Since these viruses did display neuroattenuation they could be used as a possible vaccine.

TABLE 6
Reduced Specific Infectivity and Neuroattenuation in CD155tg mice.
		Purified	Purified	Specific	PLD50	PLD50
Virus	A260	Particles/mla	PFU/ml	Infectivityb	(Particles)c	(PFU)d
PV-M(wt)	0.956	8.97 × 1012	6.0 × 1010	1/137	104.0	101.9
PV-Max	0.842	7.92 × 1012	6.0 × 1010	1/132	104.1	101.9
PV-MinXY	0.944	8.87 × 1012	9.6 × 108	1/9,200	107.1	103.2
PV-MinZ	0.731	6.87 × 1012	5.1 × 108	1/13,500	107.3	103.2
aThe A260 was used to determine particles/ml via the formula 9.4 × 1012 particles/ml = 1 OD260 unit
bCalculated by dividing the PFU/ml of purified virus by the Particles/ml
c,dcalculated after administration of virus via intracerebral injection to CD155tg mice at varying doses

Vaccination of CD155tg Mice Provides Immunity and Protection Against Lethal Challenge.

Groupings of 4-6, 6-8 week old CD155tg mice (Tg21 strain) were injected intracerebrally with purified virus dilutions from 10² particles to 10⁹ particles in 30 ul PBS to determine neuropathogenicity (Koike, et al., 1991).

The lethal dose (LD₅₀) was calculated by the Reed and Muench method (Reed and Muench, 1938). Viral titers in the spinal chord and brain were quantified by plaque assay (data not shown).

PV-MinZ and PV-MinXY encode exactly the same proteins as wild-type virus, but are attenuated in several respects, both a reduced specific infectivity and neuroattenuation.

To test PV-Min Z, PV-MinXY as a vaccine, three sub-lethal dose (10⁸ particles) of this virus was administered in 100 ul of PBS to 8, 6-8 week old CD155tg mice via intraperitoneal injection once a week for three weeks. One mouse from the vaccine cohort did not complete vaccine regimen due to illness. Also a set of control mice received three mock vaccinations with 100 ul PBS. Approximately one week after the final vaccination, 30 ul of blood was extracted from the tail vein. This blood was subjected to low speed centrifugation and serum harvested. Serum conversion against PV(M)-wt was analyzed via micro-neutralization assay with 100 plaque forming units (PFU) of challenge virus, performed according to the recommendations of WHO (Toyoda et al., 2007; Wahby, A. F., 2000). Two weeks after the final vaccination the vaccinated and control mice were challenged with a lethal dose of PV(M)-wt by intramuscular injection with a 10⁶ PFU in 100 ul of PBS (Toyoda et al., 2007). All experiments utilizing CD155tg mice were undertaken in compliance with Stony Brook University's IACUC regulations as well as federal guidelines. All 14 vaccinated mice survived and showed no signs of paralysis or parasia; in contrast, all mock-vaccinated mice died (Table 7). These data suggest that indeed the CPB virus using de-optimized codon pairs is able to immunize against the wild-type virus, providing both a robust humeral response, and also allowing complete survival following challenge.

TABLE 7
Protection Against Lethal Challenge
Virusa          Mice Protected (out of 7)b
PV-MinZ         7
PV-MinXY        7
Mock vaccinated 0
aCD155tg mice received three vaccination doses (108 particles) of respective virus
bchallenged with 106 PFU of PV(M)-wt via intramuscular injection.

Example 10

Application of SAVE to Influenza Virus

Influenza virus has 8 separate genomic segments. GenBank deposits disclosing the segment sequences for Influenza A virus (A/Puerto Rico/8/34/Mount Sinai (H1N1)) include AF389115 (segment 1, Polymerase PB2), AF389116 (segment 2, Polymerase PB1), AF389117 (segment 3, Polymerase PA), AF389118 (segment 4, hemagglutinin HA), AF389119 (segment 5, nucleoprotein NP), AF389120 (segment 6, neuraminidase NA), AF389121 (segment 7, matrix proteins M1 and M2), and AF389122 (segment 8, nonstructural protein NS1).

In initial studies, the genomic segment of strain A/PR/8/34 (also referred to herein as A/PR8) encoding the nucleoprotein NP, a major structural protein and the second most abundant protein of the virion (1,000 copies per particle) that binds as monomer to full-length viral RNAs to form coiled ribonucleoprotein, was chosen for deoptimization. (See Table 8, below, for parent and deoptimized sequences). Moreover, NP is involved in the crucial switch from mRNA to template and virion RNA synthesis (Palese and Shaw, 2007). Two synonymous encodings were synthesized, the first replacing frequently used codons with rare synonymous codons (NP^CD) (i.e., de-optimized codon bias) and, the second, de-optimizing codon pairs (NP^CPmin). The terminal 120 nucleotides at either end of the segment were not altered so as not to interfere with replication and encapsidation. NP^CD contains 338 silent mutations and NP^CPmin (SEQ ID NO:23) contains 314 silent mutations. The mutant NP segments were introduced into ambisense vectors as described (below), and together with the other seven wt influenza plasmids co-transfected into 293T/MDCK co-cultured cells. As a control, cells were transfected with all 8 wt A/PR8 plasmids. Cells transfected with the NP^CD segment and the NP^CPmin segment produced viable influenza virus similarly to cells transfected with wild-type NP. These new de-optimized viruses, referred to as A/PR8-NP^CD or A/PR8-NP^CPmin, respectively, appear to be attenuated: The titer (in terms of PFU) is 3- to 10-fold lower than the wild-type virus, and the mutant viruses both make small plaques.

Although the de-optimized influenza viruses are not as severely attenuated as a poliovirus containing a similar number of de-optimized codons, there is a difference in the translational strategies of the two viruses. Poliovirus has a single long mRNA, translated into a single polyprotein. Slow translation through the beginning of this long mRNA (as in our capsid de-optimized viruses) will reduce translation of the entire message, and thus affect all proteins. In contrast, influenza has eight separate segments, and de-optimization of one will have little if any effect on translation of the others. Moreover, expression of the NP protein is particularly favored early in influenza virus infection (Palese and Shaw, 2007).

Characterization of Influenza Virus Carrying a Codon Pair Deoptimized NP Segment

The growth characteristics of A/PR8-NP^CPmin were analyzed by infecting confluent monolayers of Madin Darby Canine Kidney cells (MDCK cells) in 100 mm dishes with 0.001 multiplicities of infection (MOI). Virus inoculums were allowed to adsorb at room temperature for 30 minutes on a rocking platform, then supplemented with 10 ml of Dulbecco Modified Eagle Medium (DMEM) containing 0.2% Bovine Serum Albumin (BSA) and 2 ug/ml TPCK treated Trypsin and incubated at 37 C. After 0, 3, 6, 9, 12, 24, and 48 hours, 100 μl of virus containing medium was removed and virus titers determined by plaque assay.

Viral titers and plaque phenotypes were determined by plaque assay on confluent monolayers of MDCK cells in 35 mm six well plates. 10-fold serial dilutions of virus were prepared in Dulbecco Modified Eagle Medium (DMEM) containing 0.2% Bovine Serum Albumin (BSA) and 2 μg/ml TPCK treated Trypsin. Virus dilutions were plated out on MDCK cells and allowed to adsorb at room temperature for 30 minutes on a rocking platform, followed by a one hour incubation at 37 C in a cell culture incubator. The inoculum was then removed and 3 ml of Minimal Eagle Medium containing 0.6% tragacanth gum (Sigma-Aldrich) 0.2% BSA and 2 ug/ml TPCK treated Trypsin. After 72 hours of incubation at 37 C, plaques were visualized by staining the wells with crystal violet.

A/PR8-NP^Min produced viable virus that produced smaller plaques on MDCK cells compared to the A/PR8 wt (FIG. 16A). Furthermore, upon low MOI infection A/PR8-NP^Min manifests a delayed growth kinetics, between 3-12 hrs post infection, where A/PR8-NP^Min titers lags 1.5 logs behind A/PR8 (FIG. 16B). Final titers are were 3-5 fold lower than that of A/PR8 (average of three different experiments).

Characterization of Influenza Viruses A/PR8-PB1^Min-RR, A/PR8-HA^Min and A/PR8-HA^Min/NP^Min Carrying Codon Pair Deoptimized PB1, HA, or HA and NP Segments.

Codon pair de-optimized genomic segments of strain A/PR/8/34 encoding the hemagglutinin protein HA and the polymerase subunit PB1 were produced. HA is a viral structural protein protruding from the viral surface mediating receptor attachment and virus entry. PB1 is a crucial component of the viral RNA replication machinery. Specifically a synonymous encoding of PB1 (SEQ ID NO:15) was synthesized by de-optimizing codon pairs between codons 190-488 (nucleotides 531-1488 of the PB1 segment) while retaining the wildtype codon usage (PB1^Min-RR). Segment PB1^Min-RR contains 236 silent mutations compared the wt PB1 segment.

A second synonymous encoding of HA (SEQ ID NO:21) was synthesized by de-optimizing codon pairs between codons 50-541 (nucleotides 180-1655 of the HA segment) while retaining the wildtype codon usage (HA^Min). HA^Min contains 355 silent mutations compared the to wt PB1 segment.

The mutant PB1^Min-RR and HA^Min segments were introduced into an ambisense vector as described above and together with the other seven wt influenza plasmids co-transfected into 293T/MDCK co-cultured cells. In addition the HA^Min segment together with the NP^Min segment and the remaining six wt plasmids were co-transfected. As a control, cells were transfected with all 8 wt A/PR8 plasmids. Cells transfected with either PB1^Min-RR or HA^Min segments produced viable virus as did the combination of the codon pair deoptimized segments HA^Min and NP^Min. The new de-optimized viruses are referred to as A/PR8-PB1^Min-RR, A/PR8-HA^Min, and A/PR8-HA^Min/NP^Min respectively.

Growth characteristics and plaque phenotypes were assessed as described above.

A/PR8-PB1^Min-RR, A/PR8-HA^Min, and A/PR8-HA^Min/NP^Min all produced viable virus. A/PR8-PB1^Min-RR and A/PR8-HA^Min/NP^Min produced smaller plaques on MDCK cells compared to the A/PR8 wt (FIG. 17A). Furthermore, upon low MOI infection on MDCK cells A/PR8-HA^Min and A/PR8-HA^Min/NP^Min display much reduced growth kinetics, especially from 3-12 hrs post infection, where A/PR8-HA^Min/NP^Min titers lag 1 to 2 orders of magnitude behind A/PR8 (FIG. 17B). Final titers for both A/PR8-HA^Min and A/PR8-HA^Min/NP^Min were 10 fold lower than that of A/PR8. As A/PR8-HA^Min/NP^Min is more severely growth retarded than A/PR8-HA^Min, it can be concluded that the effect of deoptimizing two segments is additive.

Attenuation of A/PR8-NP^Min in a BALB/c Mouse Model

Groups of 6-8 anesthetized BALB/c mice 6 weeks of age were given 12.5 μl of A/PR8 or A/PR8-NP^Min virus solution to each nostril containing 10-fold serial dilutions between 10² and 10⁶ PFU of virus. Mortality and morbidity (weight loss, reduced activity, death) was monitored. The lethal dose 50, LD₅₀, was calculated by the method of Reed and Muench (Reed, L. J., and M. Muench. 1938. Am. J. Hyg. 27:493-497).

Eight mice were vaccinated once by intranasal inoculation with 10² PFU of A/PR8-NP^Min virus. A control group of 6 mice was not vaccinated with any virus (mock). 28 days following this initial vaccination the mice were challenged with a lethal dose of the wt virus A/PR8 corresponding to 100 times the LD50.

The LD50 for A/PR8 was 4.6×10¹ PFU while the LD50 for A/PR8-NP^Min was 1×10³ PFU. At a dose of 10² all A/PR8-NP^Min infected mice survived. It can be concluded that A/PR8-NP^Min is attenuated in mice by more than 10 fold compared to the wt A/PR8 virus. This concentration was thus chosen for vaccination experiments. Vaccination of mice with 10² A/PR8-NP^Min resulted in a mild and brief illness, as indicated by a relative weight loss of less than 10% (FIG. 18A). All 8 out of 8 vaccinated mice survived. Mice infected with A/PR8 at the same dose experienced rapid weight loss with severe disease. 6 of 8 mice infected with A/PR8 died between 10 and 13 days post infection (FIG. 18B). Two mice survived and recovered from the wildtype infection.

Upon challenge with 100 times LD50 of wt virus, all A/PR8-NP^Min vaccinated were protected, and survived the challenge without disease symptoms or weight loss (FIG. 18C). Mock vaccinated mice on the other hand showed severe symptoms, and succumbed to the infection between 9 and 11 days after challenge. It can be concluded that A/PR8-NP^Min induced protective immunity in mice and, thus, has potential as a live attenuated influenza vaccine. Other viruses such as A/PR8-PB1^Min-RR and A/PR8-HA^Min/NP^Min, yet to be tested in mice, may lead to improve further the beneficial properties of codon-pair deoptimized influenza viruses as vaccines.

Example 11

Development of Higher-Throughput Methods for Making and Characterizing Viral Chimeras

Constructing Chimeric Viruses by Overlapping PCR

The “scan” through each attenuated mutant virus is performed by placing approximately 300-bp fragments from each mutant virus into a wt context using overlap PCR. Any given 300-bp segment overlaps the preceding segment by ˜200 bp, i.e., the scanning window is ˜300 bp long, but moves forward by ˜100 bp for each new chimeric virus. Thus, to scan through one mutant virus (where only the ˜3000 bp of the capsid region has been altered) requires about 30 chimeric viruses. The scan is performed in 96-well dish format which has more than sufficient capacity to analyze two viruses simultaneously.

The starting material is picogram amounts of two plasmids, one containing the sequence of the wt virus, and the other the sequence of the mutant virus. The plasmids include all the necessary elements for the PV reverse genetics system (van der Werf et al., 1986), including the T7 RNA polymerase promoter, the hammerhead ribozyme (Herold and Aldino, 2000), and the DNA-encoded poly(A) tail. Three pairs of PCR primers are used, the A, M (for Mutant), and B pairs. See FIG. 9. The M pair amplifies the desired 300 bp segment of the mutant virus; it does not amplify wt, because the M primer pairs are designed based on sequences that have been significantly altered in the mutant. The A and B pairs amplify the desired flanks of the wt viral genome Importantly, about 20-25 bp of overlap sequence is built into the 5′ ends of each M primer as well as A2 and B1, respectively; these 20-25 bps overlap (100% complementarity) with the 3′ end of the A segment and the 5′ end of the B segment, respectively.

To carry out the overlapping PCR, one 96-well dish contains wt plasmid DNA, and the 30 different A and B pairs in 30 different wells. A separate but matching 96-well plate contains mutant plasmid DNA and the 30 different M primer pairs. PCR is carried out with a highly processive, low error rate, heat-stable polymerase. After the first round of PCR, each reaction is treated with DpnI, which destroys the template plasmid by cutting at methylated GmATC sites. An aliquot from each wt and matching mutant reaction is then mixed in PCR reaction buffer in a third 96-well dish. This time, primers flanking the entire construct are used (i.e., the A1 and B2 primers). Since each segment (A, M, and B) is designed to overlap each adjacent segment by at least 20 bp, and since the reaction is being driven by primers that can only amplify a full-length product, the segments anneal and mutually extend, yielding full-length product after two or three cycles. This is a “3-tube” (three 96-well dish) design that may be compacted to a “1-tube” (one 96-well dish) design.

Characterization of Chimeric Viruses

Upon incubation with T7 RNA polymerase, the full length linear chimeric DNA genomes produced above with all needed upstream and downstream regulatory elements yields active viral RNA, which produces viral particles upon incubation in HeLa S10 cell extract (Molla et al., 1991) or upon transfection into HeLa cells. Alternatively, it is possible to transfect the DNA constructs directly into HeLa cells expressing the T7 RNA polymerase in the cytoplasm.

The functionality of each chimeric virus is then assayed using a variety of relatively high-throughput assays, including visual inspection of the cells to assess virus-induced CPE in 96-well format; estimation of virus production using an ELISA; quantitative measurement of growth kinetics of equal amounts of viral particles inoculated into cells in a series of 96-well plates; and measurement of specific infectivity (infectious units/particle [IU/P] ratio).

The functionality of each chimeric virus can then be assayed. Numerous relatively high-throughput assays are available. A first assay may be to visually inspect the cells using a microscope to look for virus-induced CPE (cell death) in 96-well format. This can also be run an automated 96-well assay using a vital dye, but visual inspection of a 96-well plate for CPE requires less than an hour of hands-on time, which is fast enough for most purposes.

Second, 3 to 4 days after transfection, virus production may be assayed using the ELISA method described in Example 3. Alternatively, the particle titer is determined using sandwich ELISA with capsid-specific antibodies. These assays allow the identification of non-viable constructs (no viral particles), poorly replicating constructs (few particles), and efficiently replicating constructs (many particles), and quantification of these effects.

Third, for a more quantitative determination, equal amounts of viral particles as determined above are inoculated into a series of fresh 96-well plates for measuring growth kinetics. At various times (0, 2, 4, 6, 8, 12, 24, 48, 72 h after infection), one 96-well plate is removed and subjected to cycles of freeze-thawing to liberate cell-associated virus. The number of viral particles produced from each construct at each time is determined by ELISA as above.

Fourth, the IU/P ratio can be measured (see Example 3).

Higher Resolution Scans

If the lethality of the viruses is due to many small defects spread through the capsid region, as the preliminary data indicate, then many or most of the chimeras are sick and only a few are non-viable. If this is the case, higher-resolution scans are probably not necessary. Conversely, if one or more of the 300 bp segments do cause lethality (as is possible for the codon-deoptimized virus in the segment between 1513 and 2470 which, as described below, may carry a translation frameshift signal that contribute to the strong phenotype of this segment), the genome scan is repeated at higher resolution, for instance a 30 bp window moving 10 bp between constructs over the 300-bp segment, followed by phenotypic analysis. A 30-bp scan does not involve PCR of the mutant virus; instead, the altered 30-bp segment is designed directly into PCR primers for the wt virus. This allows the changes responsible for lethality to be pinpointed.

Example 12

Ongoing Investigations into the Molecular Mechanisms Underlying SAVE

Choice of Chimeras

Two to four example chimeras from each of the two parental inviable viruses (i.e., 4 to 8 total viruses) are used in the following experiments. Viable chimeras having relatively small segments of mutant DNA, but having strong phenotypes are selected. For instance, viruses PV-AB^755-1513, PVAB^2470-2954 and PV-AB^2954-3386 from the deoptimized codon virus (see Example 1), and PV-Min^755-2470 and PV-Min^2470-3386 (see Example 7), are suitable. Even better starting chimeras, with smaller inserts that will make analysis easier, may also be obtained from the experiments described above (Example 8).

RNA Abundance/Stability

Conceivably the altered genome sequence destabilizes the viral RNA. Such destabilization could be a direct effect of the novel sequence, or an indirect effect of a pause in translation, or other defect in translation (see, e.g., Doma and Parker, 2006). The abundance of the mutant viral RNA is therefore examined Equal amounts of RNA from chimeric mutant virus, and wt virus are mixed and transfected into HeLa cells. Samples are taken after 2, 4, 8, and 12 h, and analyzed by Northern blotting or quantitative PCR for the two different viral RNAs, which are easily distinguishable since there are hundreds of nucleotide differences. A control with wt viral RNA compared to PV-SD (the codon-shuffled virus with a wt phenotype) is also done. A reduced ratio of mutant to wt virus RNA indicates that the chimera has a destabilized RNA.

In Vitro Translation

Translation was shown to be reduced for the codon-deoptimized virus and some of its derivatives. See Example 5. In vitro translation experiments are repeated with the codon pair-deoptimized virus (PV-Min) and its chosen chimeras. There is currently no good theory, much less any evidence, as to why deoptimized codon pairs should lead to viral inviability, and hence, investigating the effect on translation may help illuminate the underlying mechanism.

In vitro translations were performed in two kinds of extracts in Example 5. One was a “souped up” extract (Molla et al., 1991), in which even the codon-deoptimized viruses gave apparently good translation. The other was an extract more closely approximating normal in vivo conditions, in which the deoptimized-codon viruses were inefficiently translated. There were four differences between the extracts: the more “native” extract was not dialyzed; endogenous cellular mRNAs were not destroyed with micrococcal nuclease; the extract was not supplemented with exogenous amino acids; and the extract was not supplemented with exogenous tRNA. In the present study, these four parameters are altered one at a time (or in pairs, as necessary) to see which have the most significant effect on translation. For instance, a finding that it is the addition of amino acids and tRNA that allows translation of the codon-deoptimized virus strongly supports the hypothesis that translation is inefficient simply because rare aminoacyl-tRNAs are limiting. Such a finding is important from the point of view of extending the SAVE approach to other kinds of viruses.

Translational Frameshifting

Another possible defect is that codon changes could promote translational frameshifting; that is, at some codon pairs, the ribosome could shift into a different reading frame, and then arrive at an in-frame stop codon after translating a spurious peptide sequence. This type frameshifting is an important regulatory event in some viruses. The present data reveal that all PV genomes carrying the AB mutant segment from residue 1513 to 2470 are non-viable. Furthermore, all genomes carrying this mutant region produce a novel protein band during in vitro translation of approximately 42-44 kDa (see FIG. 5A, marked by asterisk). This novel protein could be the result of a frameshift.

Examination of the sequence in the 1513-2470 interval reveals three potential candidate sites that conform to the slippery heptameric consensus sequence for −1 frameshifting in eukaryotes (X-XXY-YYZ) (Farabaugh, 1996). These sites are A-AAA-AAT at positions 1885 and 1948, and T-TTA-TTT at position 2119. They are followed by stop codons in the −1 frame at 1929, 1986 or 2149, respectively. The former two seem the more likely candidates to produce a band of the observed size.

To determine whether frameshifting is occurring, each of the three candidate regions is separately mutated so that it becomes unfavorable for frameshifting. Further, each of the candidate stop codons is separately mutated to a sense codon. These six new point mutants are tested by in vitro translation. Loss of the novel 42-44 kDa protein upon mutation of the frameshifting site to an unfavorable sequence, and an increase in molecular weight of that protein band upon elimination of the stop codon, indicate that frameshifting is occurring. If frameshifting is the cause of the aberrant translation product, the viability of the new mutant that lacks the frameshift site is tested in the context of the 1513-2470 mutant segment. Clearly such a finding would be of significance for future genome designs, and if necessary, a frameshift filter may be incorporated in the software algorithm to avoid potential frameshift sites.

More detailed investigations of translational defects are conducted using various techniques including, but not limited to, polysome profiling, toeprinting, and luciferase assays of fusion proteins.

Polysome Profiling

Polysome profiling is a traditional method of examining translation. It is not high-throughput, but it is very well developed and understood. For polysome profiling, cell extracts are made in a way that arrests translation (with cycloheximide) and yet preserves the set of ribosomes that are in the act of translating their respective mRNAs (the “polysomes”). These polysomes are fractionated on a sucrose gradient, whereby messages associated with a larger number of ribosomes sediment towards the bottom. After fractionation of the gradient and analysis of RNA content using UV absorption, a polysome profile is seen where succeeding peaks of absorption correspond to mRNAs with N+1 ribosomes; typically 10 to 15 distinct peaks (representing the 40S ribosomal subunit, the 60S subunit, and 1, 2, 3, . . . 12, 13 ribosomes on a single mRNA) can be discerned before the peaks smudge together. The various fractions from the sucrose gradient are then run on a gel, blotted to a membrane, and analyzed by Northern analysis for particular mRNAs. This then shows whether that particular mRNA is primarily engaged with, say, 10 to 15 ribosomes (well translated), or 1 to 4 ribosomes (poorly translated).

In this study, for example, the wt virus, the PV-AB (codon deoptimized) virus, and its derivatives PV-AB^755-1513, and PV-AB^2954-3386, which have primarily N-terminal or C-terminal deoptimized segments, respectively, are compared. The comparison between the N-terminal and C-terminal mutant segments is particularly revealing. If codon deoptimization causes translation to be slow, or paused, then the N-terminal mutant RNA is associated with relatively few ribosomes (because the ribosomes move very slowly through the N-terminal region, preventing other ribosomes from loading, then zip through the rest of the message after traversing the deoptimized region). In contrast, the C-terminal mutant RNA are associated with a relatively large number of ribosomes, because many ribosomes are able to load, but because they are hindered near the C-terminus, they cannot get off the transcript, and the number of associated ribosomes is high.

Polysome analysis indicates how many ribosomes are actively associated with different kinds of mutant RNAs, and can, for instance, distinguish models where translation is slow from models where the ribosome actually falls off the RNA prematurely. Other kinds of models can also be tested.

Toeprinting

Toeprinting is a technique for identifying positions on an mRNA where ribosomes are slow or paused. As in polysome profiling, actively translating mRNAs are obtained, with their ribosomes frozen with cycloheximide but still associated; the mRNAs are often obtained from an in vitro translation reaction. A DNA oligonucleotide primer complementary to some relatively 3′ portion of the mRNA is used, and then extended by reverse transcriptase. The reverse transcriptase extends until it collides with a ribosome. Thus, a population of translating mRNA molecules generates a population of DNA fragments extending from the site of the primer to the nearest ribosome. If there is a site or region where ribosomes tend to pause (say, 200 bases from the primer), then this site or region will give a disproportionate number of DNA fragments (in this case, fragments 200 bases long). This then shows up as a “toeprint” (a band, or dark area) on a high resolution gel. This is a standard method for mapping ribosome pause sites (to within a few nucleotides) on mRNAs.

Chimeras with segments of deoptimized codons or codon pairs, wherein in different chimeras the segments are shifted slightly 5′ or 3′, are analyzed. If the deoptimized segments cause ribosomes to slow or pause, the toeprint shifts 5′ or 3′ to match the position of the deoptimized segment. Controls include wt viral RNA and several (harmlessly) shuffled viral RNAs. Controls also include pure mutant viral RNA (i.e., not engaged in translation) to rule out ribosome-independent effects of the novel sequence on reverse transcription.

The toeprint assay has at least two advantages. First, it can provide direct evidence for a paused ribosome. Second, it has resolution of a few nucleotides, so it can identify exactly which deoptimized codons or deoptimized codon pairs are causing the pause. That is, it may be that only a few of the deoptimized codons or codon pairs are responsible for most of the effect, and toe-printing can reveal that.

Dual Luciferase Reporter Assays of Fusion Proteins

The above experiments may suggest that certain codons or codon pairs are particularly detrimental for translation. As a high-throughput way to analyze effects of particular codons and codon pairs on translation, small test peptides are designed and fused to the N-terminus of sea pansy luciferase. Luciferase activity is then measured as an assay of the translatability of the peptide. That is, if the N-terminal peptide is translated poorly, little luciferase will be produced.

A series of eight 25-mer peptides are designed based on the experiments above. Each of the eight 25-mers is encoded 12 different ways, using various permutations of rare codons and/or rare codon pairs of interest. Using assembly PCR, these 96 constructs (8 25-mers×12 encodings) are fused to the N-terminus of firefly luciferase (F-luc) in a dicistronic, dual luciferase vector described above (see Example 5 and FIG. 6). A dual luciferase system uses both the firefly luciferase (F-Luc) and the sea pansy (Renilla) luciferase (R-Luc); these emit light under different biochemical conditions, and so can be separately assayed from a single tube or well. A dicistronic reporter is expressed as a single mRNA, but the control luciferase (R-Luc) is translated from one internal ribosome entry site (IRES), while the experimental luciferase (F-luc) (which has the test peptides fused to its N-terminus) is independently translated from its own IRES. Thus, the ratio of F-Luc activity to R-Luc activity is an indication of the translatability of the test peptide. See FIG. 6.

The resulting 96 dicistronic reporter constructs are transfected directly from the PCR reactions into 96 wells of HEK293 or HeLa cells. The firefly luciferase of the upstream cistron serves as an internal transfection control. Codon- or codon-pair-dependent expression of the sea pansy luciferase in the second cistron can be accurately determined as the ratio between R-Luc and F-Luc. This assay is high-throughput in nature, and hundreds or even thousands of test sequences can be assayed, as necessary.

Example 13

Design and Synthesis of Attenuated Viruses Using Novel Alternative-Codon Strategy

The SAVE approach to re-engineering viruses for vaccine production depends on large-scale synonymous codon substitution to reduce translation of viral proteins. This can be achieved by appropriately modulating the codon and codon pair bias, as well as other parameters such as RNA secondary structure and CpG content. Of the four de novo PV designs, two (the shuffled codon virus, PV-SD, and the favored codon pair virus, PV-Max) resulted in little phenotypic change over the wt virus. The other two de novo designs (PV-AB and PV-Min) succeeded in killing the virus employing only synonymous substitutions through two different mechanisms (drastic changes in codon bias and codon pair bias, respectively). The live-but-attenuated strains were constructed by subcloning regions from the inactivated virus strains into the wt.

A better understanding of the underlying mechanisms of viral attenuation employing large scale synonymous substitutions facilitates predictions of the phenotype and expression level of a synthetic virus. Ongoing studies address questions relating to the effect of the total number of alterations or the density of alterations on translation efficiency; the effect of the position of dense regions on translation; the interaction of codon and codon pair bias; and the effect of engineering large numbers of short-range RNA secondary structures into the genome. It is likely that there is a continuum between the wt and inactivated strains, and that any desired attenuation level can be engineered into a weakened strain. However, there may be hard limits on the attenuation level that can be achieved for any infection to be at self-sustaining and hence detectable. The 15⁴⁴² encodings of PV proteins constitutes a huge sequence space to explore, and various approaches are being utilized to explore this sequence space more systematically. These approaches include, first, developing a software platform to help design novel attenuated viruses, and second, using this software to design, and then synthesize and characterize, numerous new viruses that explore more of the sequence space, and answer specific questions about how alternative encodings cause attenuation. Additionally, an important issue to consider is whether dangerous viruses might accidentally be created by apparently harmless shuffling of synonymous codons.

Development of Software for Computer-Based Design of Viral Genomes and Data Analysis

Designing synthetic viruses requires substantial software support for (1) optimizing codon and codon-pair usage and monitoring RNA secondary structure while preserving, embedding, or removing sequence specific signals, and (2) partitioning the sequence into oligonucleotides that ensure accurate sequence-assembly. The prototype synthetic genome design software tools are being expanded into a full environment for synthetic genome design. In this expanded software, the gene editor is conceptually built around constraints instead of sequences. The gene designer works on the level of specifying characteristics of the desired gene (e.g., amino acid sequence, codon/codon-pair distribution, distribution of restriction sites, and RNA secondary structure constraints), and the gene editor algorithmically designs a DNA sequence realizing these constraints. There are many constraints, often interacting with each other, including, but not limited to, amino acid sequence, codon bias, codon pair bias, CG dinucleotide content, RNA secondary structure, cis-acting nucleic acid signals such as the CRE, splice sites, polyadenylation sites, and restriction enzyme recognition sites. The gene designer recognizes the existence of these constraints, and designs genes with the desired features while automatically satisfying all constraints to a pre-specified level.

The synthesis algorithms previously developed for embedding/removing patterns, secondary structures, overlapping coding frames, and adhering to codon/codon-pair distributions are implemented as part of the editor, but more important are algorithms for realizing heterogeneous combinations of such preferences. Because such combinations lead to computationally intractable (NP-complete) problems, heuristic optimization necessarily plays an important role in the editor. Simulated annealing techniques are employed to realize such designs; this is particularly appropriate as simulated annealing achieved its first practical use in the early VLSI design tools.

The full-featured gene design programming environment is platform independent, running in Linux, Windows and MacOS. The system is designed to work with genomes on a bacterial or fungal (yeast) scale, and is validated through the synthesis and evaluation of the novel attenuated viral designs described below.

Virus Designs with Extreme Codon Bias in One or a Few Amino Acids

For a live vaccine, a virus should be as debilitated as possible, short of being inactivated, in which case there is no way to grow and manufacture the virus. One way of obtaining an optimally debilitated is to engineer the substitution of rare codons for just one or a few amino acids, and to create a corresponding cell line that overexpresses the rare tRNAs that bind to those rare codons. The virus is then able to grow efficiently in the special, permissive cell line, but is inviable in normal host cell lines. Virus is grown and manufactured using the permissive cell line, which is not only very convenient, but also safer than methods used currently used for producing live attenuated vaccines.

With the sequencing of the human genome, information regarding copy number of the various tRNA genes that read rare codons is available. Based on the literature (e.g., Lavner and Kotlar, 2005), the best rare codons for present purposes are CTA (Leu), a very rare codon which has just two copies of the cognate tRNA gene; TCG (Ser), a rare codon with four copies of the cognate tRNA gene; and CCG (Pro), a rare codon with four copies of the cognate tRNA gene (Lavner and Kotlar, 2005). The median number of copies for a tRNA gene of a particular type is 9, while the range is 2 to 33 copies (Lavner and Kotlar, 2005). Thus, the CTA codon is not just a rare codon, but is also the one codon with the fewest cognate tRNA genes. These codons are not read by any other tRNA; for instance, they are not read via wobble base pairing.

Changing all the codons throughout the virus genome coding for Leu (180 codons), Ser (153), and Pro (119) to the rare synonymous codons CTA, TCG, or CCG, respectively, is expected to create severely debilitated or even non-viable viruses. Helper cells that overexpress the corresponding rare tRNAs can then be created. The corresponding virus is absolutely dependent on growing only in this artificial culture system, providing the ultimate in safety for the generation of virus for vaccine production.

Four high-priority viruses are designed and synthesized: all Leu codons switched to CTA; all Ser codons switched to TCG; all Pro codons switched to CCG; and all Leu, Ser, and Pro codons switched to CTA, TCG, and CCG, respectively, in a single virus. In one embodiment, these substitutions are made only in the capsid region of the virus, where a high rate of translation is most important. In another embodiment, the substitutions are made throughout the virus.

CG Dinucleotide Bias Viruses

With few exceptions, virus genomes under-represent the dinucleotide CpG, but not GpC (Karlin et al., 1994). This phenomenon is independent of the overall G+C content of the genome. CpG is usually methylated in the human genome, so that single-stranded DNA containing non-methylated CpG dinucleotides, as often present in bacteria and DNA viruses, are recognized as a pathogen signature by the Toll-like receptor 9. This leads to activation of the innate immune system. Although a similar system has not been shown to operate for RNA viruses, inspection of the PV genome suggests that PV has selected against synonymous codons containing CpG to an even greater extent than the significant under-representation of CpG dinucleotides in humans. This is particularly striking for arginine codons. Of the six synonymous Arg codons, the four CG containing codons (CGA, CGC CGG, CCU) together account for only 24 of all 96 Arg codons while the remaining two (AGA, AGG) account for 72. This in contrast to the average human codon usage, which would predict 65 CG containing codons and 31 AGA/AGO codons. In fact, two of the codons under-represented in PV are frequently used in human cells (CGC, CGG). There are two other hints that CG may be a disadvantageous dinucleotide in PV. First, in the codon pair-deoptimized virus, many of the introduced rare codon pairs contain CG as the central dinucleotide of the codon pair hexamer. Second, when Burns et al. (2006) passaged their codon bias-deoptimized virus and sequenced the genomes, it was observed that these viruses evolved to remove some CG dinucleotides.

Thus, in one high-priority redesigned virus, most or all Arg codons are changed to CGC or CGG (two frequent human codons). This does not negatively affect translation and allows an assessment of the effect of the CpG dinucleotide bias on virus growth. The increased C+G content of the resulting virus requires careful monitoring of secondary structure; that is, changes in Arg codons are not allowed to create pronounced secondary structures.

Modulating Codon-Bias and Codon-Pair Bias Simultaneously.

Codon bias and codon-pair bias could conceivably interact with each other at the translational level. Understand this interaction may enable predictably regulation of the translatability of any given protein, possibly over an extreme range.

If we represent wild type polio codon bias and codon pair bias as 0, and the worst possible codon bias and codon pair bias as −1, then four high-priority viruses are the (−0.3, −0.3), (−0.3, −0.6), (−0.6, −0.3), and (−0.6, −0.6) viruses. These viruses reveal how moderately poor or very poor codon bias interacts with moderately poor or very poor codon pair virus. These viruses are compared to the wild type and also to the extreme PV-AB (−1, 0) and PV-Min (0, −1) designs.

Modulating RNA Secondary Structure

The above synthetic designs guard against excessive secondary structures. Two additional designs systematically avoid secondary structures. These viruses are engineered to have wt codon and codon-pair bias with (1) provably minimal secondary structure, and (2) many small secondary structures sufficient to slow translation.

Additional Viral Designs

Additional viral designs include full-genome codon bias and codon-pair bias designs; non-CG codon pair bias designs; reduced density rare codon designs; and viruses with about 150 rare codons, either spread through the capsid region, or grouped at the N-terminal end of the capsid, or grouped at the C-terminal end of the capsid.

Example 14

Testing the Potential for Accidentally Creating Viruses of Increased Virulence

It is theoretically possible that redesigning of viral genomes with the aim of attenuating these viruses could accidentally make a virus more virulent than the wt virus. Because protein sequences are not altered in the SAVE procedure, this outcome is unlikely. Nevertheless, it is desirable to experimentally demonstrate that the SAVE approach is benign.

Out of the possible 10⁴⁴² sequences that could possibly encode PV proteins, some reasonably fit version of PV likely arose at some point in the past, and evolved to a local optimum (as opposed to a global optimum). The creation of a new version of PV with the same protein coding capacity but a very different set of codons places this new virus in a different location on the global fitness landscape, which could conceivably be close to a different local optimum than wt PV. Conceivably, this new local optimum could be better than the wild type local optimum. Thus, it is just barely possible that shuffling synonymous codons might create a fitter virus.

To investigate this possibility, 13 PV genomes are redesigned and synthesized: one virus with the best possible codon bias; one virus with the best possible codon pair bias (i.e., PV-Max); one virus with the best possible codon and codon pair bias; and 10 additional viruses with wt codon and codon pair bias, but shuffled synonymous codons. Other parameters, such as secondary structure, C+G content, and CG dinucleotide content are held as closely as possible to wt levels.

These 13 viruses may each be in a very different location of the global fitness landscape from each other and from the wild type. But none of them is at a local optimum because they have not been subject to selection. The 13 viruses and the wt are passaged, and samples viruses are taken at the 1^st, 10^th, 20^th, and 50^th passages. Their fitness is compared to each other and to wt by assessing plaque size, plaque-forming units/ml in one-step growth curves, and numbers of particles formed per cell. See Example 1. Five examples of each of the 13 viruses are sequenced after the 10^th, 20^th, and 50^th passage. Select passage isolates are tested for pathogenicity in CD155tg mice, and LD₅₀'s are determined. These assays reveal whether any of the viruses are fitter than wt, and provide a quantitative measure of the risk of accidental production of especially virulent viruses. The 10 viruses with wt levels of codon and codon pair bias also provide information on the variability of the fitness landscape at the encoding level.

In view of the possibility that a fitter virus could emerge, and that a fitter virus may be a more dangerous virus, these experiments are conducted in a BSL3 laboratory. After the 10^th passage, phenotypes and sequences are evaluated and the susceptibility of emerging viruses to neutralization by PV-specific antibodies is verified. The experiment is stopped and reconsidered if any evidence of evolution towards a significantly fitter virus, or of systematic evolution towards new protein sequences that evade antibody neutralization, is obtained. Phenotypes and sequences are similarly evaluated after passage 20 before proceeding to passage 50. Because the synthetic viruses are created to encode exactly the same proteins as wt virus, the scope for increased virulence seems very limited, even if evolution towards (slightly) increased fitness is observed.

Example 15

Extension of SAVE Approach to Virus Systems Other than Poliovirus

Notwithstanding the potential need for a new polio vaccine to combat the potential of reversion in the closing stages of the global effort at polio eradication, PV has been selected in the present studies primarily as a model system for developing SAVE. SAVE has, however, been developed with the expectation that this approach can be extended to other viruses where vaccines are needed. This extension of the SAVE strategy is herein exemplified by application to Rhinovirus, the causative agent of the common cold, and to influenza virus.

Adaptation of SAVE to Human Rhinovirus—a Virus Closely Related to Polio Virus

Two model rhinoviruses, HRV2 and HRV14, were selected to test the SAVE approach for several reasons: (1) HRV2 and HRV14 represent two members of the two different genetic subgroups, A and B (Ledford et al., 2004); (2) these two model viruses use different receptors, LDL-receptor and ICAM-1, respectively (Greve et al., 1989; Hofer et al., 1994); both viruses as well as their infectious cDNA clones have been used extensively, and most applicable materials and methods have been established (Altmeyer et al., 1991; Gerber et al., 2001); and (4) much of the available molecular knowledge of rhinoviruses stems from studies of these two serotypes.

The most promising SAVE strategies developed through the PV experiments are applied to the genomes of HRV2 and HRV14. For example, codons, codon pairs, secondary structures, or combinations thereof, are deoptimized. Two to three genomes with varying degrees of attenuation are synthesized for each genotype. Care is taken not to alter the CRE, a critical RNA secondary structure of about 60 nucleotides (Gerber et al., 2001; Goodfellow et al., 2000; McKnight, 2003). This element is vital to the replication of picornaviruses and thus the structure itself must be maintained when redesigning genomes. The location of the CRE within the genome varies for different picornaviruses, but is known for most families (Gerber et al., 2001; Goodfellow et al., 2000; McKnight, 2003), and can be deduced by homology modeling for others where experimental evidence is lacking. In the case of HRV2 the CRE is located in the RNA sequence corresponding to the nonstructural protein 2A^pro; and the CRE of HRV14 is located in the VP1 capsid protein region (Gerber et al., 2001; McKnight, 2003).

The reverse genetics system to derive rhinoviruses from DNA genome equivalents is essentially the same as described above for PV, with the exception that transfections are done in HeLa-H1 cells at 34° C. in Hepes-buffered culture medium containing 3 mM Mg++ to stabilize the viral capsid. The resulting synthetic viruses are assayed in tissue culture to determine the PFU/IU ratio. See Example 3. Plaque size and kinetics in one-step growth curves are also assayed as described. See Example 2. Because the SAVE process can be applied relatively cheaply to all 100 or so relevant rhinoviruses, it is feasible to produce a safe and effective vaccine for the common cold using this approach.

Adaptation of SAVE to Influenza A Virus—a Virus Unrelated to Poliovirus

The most promising SAVE design criteria identified from PV experimentation are used to synthesize codon-deoptimized versions of influenza virus. The influenza virus is a “segmented” virus consisting of eight separate segments of RNA; each of these can be codon-modified. The well established ambisense plasmid reverse genetics system is used for generating variants of influenza virus strain A/PR/8/34. This eight-plasmid system is a variation of what has been described previously (Hoffmann et al., 2000), and has been kindly provided by Drs. P. Palese and A. Garcia-Sastre. Briefly, the eight genome segments of influenza each contained in a separate plasmid are flanked by a Pol I promoter at the 3′ end and Pol I terminator at the 5′ end on the antisense strand. This cassette in turn is flanked by a cytomegalovirus promoter (a Pol II promoter) at the 5′ end and a polyadenylation signal at the 3′ end on the forward strand (Hoffmann et al., 2000). Upon co-transfection into co-cultured 293T and MDCK cells, each ambisense expression cassette produces two kinds of RNA molecules. The Pol II transcription units on the forward strand produce all influenza mRNAs necessary for protein synthesis of viral proteins. The Pol I transcription unit on the reverse strand produces (−) sense genome RNA segments necessary for assembly of ribonucleoprotein complexes and encapsidation. Thus, infectious influenza A/PR/8/34 particles are formed (FIG. 10). This particular strain of the H1 N1 serotype is relatively benign to humans. It has been adapted for growth in tissue culture cells and is particularly useful for studying pathogenesis, as it is pathogenic in BALB/c mice.

When synthesizing segments that are alternatively spliced (NS and M), care is taken not to destroy splice sites and the alternative reading frames. In all cases the terminal 120 nt at either end of each segment are excluded, as these sequences are known to contain signals for RNA replication and virus assembly. At least two versions of each fragment are synthesized (moderate and maximal deoptimization). Viruses in which only one segment is modified are generated, the effect is assessed, and more modified segments are introduced as needed. This is easy in this system, since each segment is on a separate plasmid.

Virus infectivity is titered by plaque assay on MDCK cells in the presence of 1 ug/ml (TPCK)-trypsin. Alternatively, depending on the number of different virus constructs, a 96-well ELISA is used to determine the titer of various viruses as cell infectious units on MDCK cells essentially as described above for PV. See Example 3. The only difference is that now a HA-specific antibody is used to stain infected cells. In addition, the relative concentration of virions are determined via hemagglutination (HA) assay using chicken red blood cells (RBC) (Charles River Laboratories) using standard protocols (Kendal et al., 1982). Briefly, virus suspensions are 2-fold serially diluted in PBS in a V-bottom 96 well plates. PBS alone is used as an assay control. A standardized amount of RBCs is added to each well, and the plates are briefly agitated and incubated at room temperature for 30 minutes. HA titers are read as the reciprocal dilution of virus in the last well with complete hemagglutination. While HA-titer is a direct corollary of the amount of particles present, PFU-titer is a functional measure of infectivity. By determining both measures, a relative PFU/HA-unit ratio is calculated similar to the PFU/particle ratio described in the PV experiments. See Example 3. This addresses the question whether codon- and codon pair-deoptimized influenza viruses also display a lower PFU/particle as observed for PV.

Virulence Test

The lethal dose 50 (LD₅₀) of the parental NPR/8/34 virus is first determined for mice and synthetic influenza viruses are chosen for infection of BALB/c mice by intranasal infection. Methods for determining LD₅₀ values are well known to persons of ordinary skill in the art (see Reed and Muench, 1938, and Example 4). The ideal candidate viruses display a low infectivity (low PFU titer) with a high virion concentration (high HA-titer). Anesthetized mice are administered 25 μl of virus solution in PBS to each nostril containing 10-fold serial dilutions between 10² to 10⁷ PFU of virus. Mortality and morbidity (weight loss, reduced activity) are monitored twice daily for up to three weeks. LD₅₀ is calculated by the method of Reed and Muench (1938). For the A/PR/8/34 wt virus the expected LD₅₀ is around 10³ PFU (Talon et al., 2000), but may vary depending on the particular laboratory conditions under which the virus is titered.

Adaptation of SAVE to Dengue, HIV, Rotavirus, and SARS

Several viruses were selected to further test the SAVE approach. Table 8 identifies the coding regions of each of Dengue, HIV, Rotavirus (two segments), and SARS, and provides nucleotide sequences for parent viruses and exemplary viral genome sequences having deoptimized codon pair bias. As described above, codon pair bias is determined for a coding sequence, even though only a portion (subsequence) may contain the deoptimizing mutations.

TABLE 8
Nucleotide sequence and codon pair bias
of parent and codon pair bias-reduced coding regions
	Parent	Codon pair bias-reduced
	sequence	sequence
	SEQ			SEQ
	ID			ID	deoptimized
Virus	NO:	CDS	CPB	NO:	segment*	CPB*
Flu PB1	13	25-2298	0.0415	14	531-2143	−0.2582
Flu PB1-	″	″	″	15	531-1488	−0.1266
RR
Flu PB2	16	28-2307	0.0054	17	33-2301	−0.3718
Flu PA	18	25-2175	0.0247	19	30-2171	−0.3814
Flu HA	20	33-1730	0.0184	21	180-1655	−0.3627
Flu NP	22	46-1542	0.0069	23	126-1425	−0.3737
Flu NA	24	21-1385	0.0037	25	123-1292	−0.3686
Flu M	26		0.0024
Flu NS	27	27-719	−0.0036	28	128-479	−0.1864
Rhino-	29	619-7113	0.051	30		−0.367
virus
89
Rhino-	31	629-7168	0.046	32		−0.418
virus
14
Dengue	33	95-10273	0.0314	34		−0.4835
HIV	35	336-1634	0.0656	36		−0.3544
		1841-4585
		4644-5102
		5858-7924
		8343-8963
Rotavirus	37	12-3284	0.0430	38		−0.2064
Seg. 1
Rotavirus	39	37-2691	0.0375	40		−0.2208
Seg. 2
SARS	41	265-13398	0.0286	42		−0.4393
		13416-21485
		21492-25259
		26398-27063
*CPB can be reduced by deoptimizing an internal segment smaller than the complete coding sequence. Nevertheless, CPB is calculated for the complete CDS.

Example 16

Assessment of Poliovirus and Influenza Virus Vaccine Candidates in Mice

The ability of deoptimized viruses to vaccinate mice against polio or influenza is tested.

Poliovirus Immunizations, Antibody Titers, and Wt Challenge Experiments

The working hypothesis is that a good vaccine candidate combines a low infectivity titer with a high virion titer. This ensures that a high amount of virus particles (i.e., antigen) can be injected while at the same time having a low risk profile. Thus, groups of five CD155tg mice will be injected intraperitoneally with 10³, 10⁴, 10⁵, and 10⁶ PFU of PV(Mahoney) (i.e., wild-type), PV1 Sabin vaccine strain, PV^AB2470-2954, PV-Min^755-2470, or other promising attenuated polioviruses developed during this study. For the wild-type, 1 PFU is about 100 viral particles, while for the attenuated viruses, 1 PFU is roughly 5,000 to 100,000 particles. Thus, injection with equal number of PFUs means that 50 to 1000-fold more particles of attenuated virus are being injected. For wt virus injected intraperitoneally, the LD₅₀ is about 10⁶ PFU, or about 10⁸ particles. Accordingly, some killing is expected with the highest doses but not with the lower doses.

Booster shots of the same dose are given one week after and four weeks after the initial inoculation. One week following the second booster, PV-neutralizing antibody titers are determined by plaque reduction assay. For this purpose, 100 PFU of wt PV(M) virus are incubated with 2-fold serial dilutions of sera from immunized mice. The residual number of PFU is determined by plaque assays. The neutralizing antibody titer is expressed as the reciprocal of the lowest serum dilution at which no plaques are observed.

Four weeks after the last booster, immunized mice and non-immunized controls are challenged with a lethal dose of PV(M) wt virus (10⁶ PFU intraperitoneally; this equals 100 times LD₅₀, and survival is monitored.

Influenza Immunizations, Antibody Titers, and Wt Challenge Experiments

For vaccination experiments, groups of 5 BALB/c mice are injected with wt and attenuated influenza viruses intraperitoneally at a dose of 0.001, 0.01, 0.1, and 1.0 LD₅₀. Booster vaccinations are given at the same intervals described above for PV. Influenza antibody titers one week after the second booster are determined by an inhibition of hemagglutination (HI) assay following standard protocols (Kendal et al., 1982). Briefly, sera from immunized and control mice treated with receptor destroying enzyme (RDE; Sigma, St Louis, Mo.) are 2-fold serially diluted and mixed with 5 HA-units of A/PR/8/34 virus in V-bottom 96 wells. RBCs are then added and plates are processed as above for the standard HA-assay. Antibody titers are expressed as the reciprocal dilution that results in complete inhibition of hemagglutination.

Three weeks after the last booster vaccination, mice are challenged infra-nasally with 100 or 1000 LD₅₀ of A/PR/8/34 parental virus (approximately 10⁵ and 10⁶ PFU), and survival is monitored.

Animal Handling

Transgenic mice expressing the human poliovirus receptor CD155 (CD155tg) were obtained from Dr. Nomoto, The Tokyo University. The CD155tg mouse colony is maintained by the State University of New York (SUNY) animal facility. BALB/c mice are obtained from Taconic (Germantown, N.Y.). Anesthetized mice are inoculated using 25-gauge hypodermic needles with 30 μl of viral suspension by intravenous, intraperitoneal or intracerebral route or 50 ul by the intranasal route. Mice of both sexes between 6-24 weeks of age are used. Mice are the most economical model system for poliovirus and influenza virus research. In addition, in the case of PV, the CD155tg mouse line is the only animal model except for non-human primates. Mice also provide the safest animal model since no virus spread occurs between animals for both poliovirus and influenza virus.

All mice are housed in SUNY's state of the art animal facility under the auspices of the Department of Laboratory Animal Research (DLAR) and its veterinary staff. All animals are checked twice weekly by the veterinary staff. Virus-infected animals are checked twice daily by the investigators and daily by the veterinary staff. All infection experiments are carried out in specially designated maximum isolation rooms within the animal facility. After conclusion of an experiment, surviving mice are euthanized and cadavers are sterilized by autoclaving. No mouse leaves the virus room alive.

In the present study, mice are not subjected to any surgical procedure besides intravenous, intracerebral, intraperitoneal, intramuscular or intranasal inoculation, the injection of anesthetics, and the collection of blood samples. For vaccination experiments, blood samples are taken prior and after vaccination for detection of virus-specific antibodies. To this end, 50-100 μl are collected from mice the day before injection and one week following the second booster vaccination. A maximum of two blood samples on individual animals are collected at least four weeks apart. Animals are anesthetized and a sharp scalpel is used to cut off 2 mm of tail. Blood is collected with a capillary tube. Subsequent sampling is obtained by removing scab on the tail. If the tail is healed, a new 2-mm snip of tail is repeated.

All animal experiments are carried out following protocols approved by the SUNY Institutional Animal Care and Use Committee (IACUC). Euthanasia is performed by trained personnel in a CO₂ gas chamber according to the recommendation of the American Veterinary Medical Association. Infection experiments are conducted under the latest the ABSL 2/polio recommendations issued by the Centers for Disease Control and Prevention (CDC).

Example 17

Codon Pair Bias Algorithm—Codon Pair Bias and Score Matrix

In most organisms, there exists a distinct codon bias, which describes the preferences of amino acids being encoded by particular codons more often than others. It is widely believed that codon bias is connected to protein translation rates. In addition, each species has specific preferences as to whether a given pair of codons appear as neighbors in gene sequences, something that is called codon-pair bias.

To quantify codon pair bias, we define a codon pair distance as the log ratio of the observed over the expected number of occurrences (frequency) of codon pairs in the genes of an organism. Although the calculation of the observed frequency of codon pairs in a set of genes is straightforward, the expected frequency of a codon pair is calculated as in Gutman and Hatfield, Proc. Natl. Acad. Sci. USA, 86:3699-3703, 1989, and is independent of amino acid and codon bias. To achieve that, the expected frequency is calculated based on the relative proportion of the number of times an amino acid is encoded by a specific codon. In short:

$\mathrm{codon}\mathrm{pair}\mathrm{score}=\log \left(\frac{F\left(\mathrm{AB}\right)}{\frac{F\left(A\right)\times F\left(B\right)}{F\left(X\right)\times F\left(Y\right)}\times F\left(\mathrm{XY}\right)}\right),$ where the codon pair AB encodes for amino acid pair XY and F denotes frequency (number of occurrences).

In this scheme we can define a 64×64 codon-pair distance matrix with all the pairwise costs as defined above. Any m-residue protein can be rated as using over- or under-represented codon pairs by the average of the codon pair scores that comprise its encoding.

Optimization of a Gene Encoding Based on Codon Pair Bias

To examine the effects of codon pair bias on the translation of specific proteins, we decided to change the codon pairs while keeping the same codon distribution. So we define the following problem: Given an amino acid sequence and a set of codon frequencies (codon distribution), change the DNA encoding of the sequence such that the codon pair score is optimized (usually minimized or maximized).

Our problem, as defined above, can be associated with the Traveling Salesman Problem (TSP). The traveling salesman problem is the most notorious NP-complete problem. This is a function of its general usefulness, and because it is easy to explain to the public at large. Imagine a traveling salesman who has to visit each of a given set of cities by car. What is the shortest route that will enable him to do so and return home, thus minimizing his total driving?

TSP Heuristics

Almost any flavor of TSP is going to be NP-complete, so the right way to proceed is with heuristics. These are often quite successful, typically coming within a few percent of the optimal solution, which is close enough for most applications and in particular for our optimized encoding.

Minimum spanning trees—A simple and popular heuristic, especially when the sites represent points in the plane, is based on the minimum spanning tree of the points. By doing a depth-first search of this tree, we walk over each edge of the tree exactly twice, once going down when we discover the new vertex and once going up when we backtrack. We can then define a tour of the vertices according to the order in which they were discovered and use the shortest path between each neighboring pair of vertices in this order to connect them. This path must be a single edge if the graph is complete and obeys the triangle inequality, as with points in the plane. The resulting tour is always at most twice the length of the minimum TSP tour. In practice, it is usually better, typically 15% to 20% over optimal. Further, the time of the algorithm is bounded by that of computing the minimum spanning tree, only O(n lg n) in the case of points in the plane.

Incremental insertion methods—A different class of heuristics inserts new points into a partial tour one at a time (starting from a single vertex) until the tour is complete. The version of this heuristic that seems to work best is furthest point insertion: of all remaining points, insert the point v into partial tour T such that

$\underset{v\in V}{\max }\stackrel{T}{\underset{t=1}{\min }}\left(d\left(v,v_i\right)+d\left(v,v_{i+1}\right)\right).$ The minimum ensures that we insert the vertex in the position that adds the smallest amount of distance to the tour, while the maximum ensures that we pick the worst such vertex first. This seems to work well because it first “roughs out” a partial tour before filling in details. Typically, such tours are only 5% to 10% longer than optimal.

k-optimal tours—Substantially more powerful are the Kernighan-Lin, or k-opt class of heuristics. Starting from an arbitrary tour, the method applies local refinements to the tour in the hopes of improving it. In particular, subsets of k≥2 edges are deleted from the tour and the k remaining subchains rewired in a different way to see if the resulting tour is an improvement. A tour is k-optimal when no subset of k edges can be deleted and rewired so as to reduce the cost of the tour. Extensive experiments suggest that 3 optimal tours are usually within a few percent of the cost of optimal tours. For k>3, the computation time increases considerably faster than solution quality. Two-opting a tour is a fast and effective way to improve any other heuristic. Simulated annealing provides an alternate mechanism to employ edge flips to improve heuristic tours.

Algorithm for Solving the Optimum Encoding Problem

Our problem as defined is associated with the problem of finding a traveling salesman path (not tour) under a 64-country metric. In this formulation, each of the 64 possible codons is analogous to a country, and the codon multiplicity modeled as the number of cities in the country. The codon-pair bias measure is reflected as the country distance matrix.

The real biological problem of the design of genes encoding specific proteins using a given set of codon multiplicities so as to optimize the gene/DNA sequence under a codon-pair bias measure is slightly different. What is missing in our model in the country TSP model is the need to encode specific protein sequences. The DNA triplet code partitions the 64 codons into 21 equivalence classes (coding for each of the 20 possible amino acids and a stop symbol). Any given protein/amino acid sequence can be specified by picking an arbitrary representative of the associated codon equivalence class to encode it.

Because of the special restrictions and the nature of our problem, as well as its adaptability to application of additional criteria in the optimization, we selected the Simulated annealing heuristic to optimize sequences. The technique is summarized below.

Simulated Annealing Heuristic

Simulated annealing is a heuristic search procedure that allows occasional transitions leading to more expensive (and hence inferior) solutions. This may not sound like a win, but it serves to help keep our search from getting stuck in local optima.

The inspiration for simulated annealing comes from the physical process of cooling molten materials down to the solid state. In thermodynamic theory, the energy state of a system is described by the energy state of each of the particles constituting it. The energy state of each particle jumps about randomly, with such transitions governed by the temperature of the system. In particular, the probability P(e_i, e_j, T) of transition from energy e_i to e_j at temperature T is given by:P(e_i,e_j,T)=e^{(ei−ej)/(kBT)} where kB is a constant, called Boltzmann's constant. What does this formula mean? Consider the value of the exponent under different conditions. The probability of moving from a high-energy state to a lower-energy state is very high. However, there is also a nonzero probability of accepting a transition into a high-energy state, with small energy jumps much more likely than big ones. The higher the temperature, the more likely such energy jumps will occur.

What relevance does this have for combinatorial optimization? A physical system, as it cools, seeks to go to a minimum-energy state. For any discrete set of particles, minimizing the total energy is a combinatorial optimization problem. Through random transitions generated according to the above probability distribution, we can simulate the physics to solve arbitrary combinatorial optimization problems.

As with local search, the problem representation includes both a representation of the solution space and an appropriate and easily computable cost function C(s) measuring the quality of a given solution. The new component is the cooling schedule, whose parameters govern how likely we are to accept a bad transition as a function of time.

At the beginning of the search, we are eager to use randomness to explore the search space widely, so the probability of accepting a negative transition should be high. As the search progresses, we seek to limit transitions to local improvements and optimizations. The cooling schedule can be regulated by the following parameters:

Initial system temperature—Typically t₁=1.

Temperature decrement function—Typically t_k=α·tk−1, where 0.8≤α≤0.99. This implies an exponential decay in the temperature, as opposed to a linear decay.

Number of iterations between temperature change—Typically, 100 to 1,000 iterations might be permitted before lowering the temperature.

Acceptance criteria—A typical criterion is to accept any transition from s_i to s_i+1 when C(s_i+1)<C(s_i) and to accept a negative transition whenever

$e^{-\frac{\left(C\left(s_i\right)-C\left(s_{i+1}\right)\right)}{{\mathrm{cs}}_i}}\ge r,$ where r is a random number 0≤r<1. The constant c normalizes this cost function, so that almost all transitions are accepted at the starting temperature.

Stop criteria—Typically, when the value of the current solution has not changed or improved within the last iteration or so, the search is terminated and the current solution reported.

In expert hands, the best problem-specific heuristics for TSP can slightly outperform simulated annealing, but the simulated annealing solution works easily and admirably.

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				Observed/
AA pair	Codon pair	Expected	Observed	Expected	CPS
AA	GCGGCG	630.04	2870	4.555	1.516
AA	GCGGCC	2330.20	4032	1.730	0.548
AA	GCTGCT	3727.41	5562	1.492	0.400
AA	GCAGCA	2856.40	4196	1.469	0.385
AA	GCAGCT	3262.97	4711	1.444	0.367
AA	GCTGCA	3262.97	4357	1.335	0.289
AA	GCTGCC	5667.77	7014	1.238	0.213
AA	GCAGCC	4961.56	6033	1.216	0.196
AA	GCAGCG	1341.51	1420	1.059	0.057
AA	GCTGCG	1532.46	1533	1.000	0.000
AA	GCGGCT	1532.46	1472	0.961	−0.040
AA	GCCGCG	2330.20	2042	0.876	−0.132
AA	GCGGCA	1341.51	1142	0.851	−0.161
AA	GCCGCC	8618.21	5141	0.597	−0.517
AA	GCCGCT	5667.77	1378	0.243	−1.414
AA	GCCGCA	4961.56	1122	0.226	−1.487
AC	GCCTGC	2333.61	3975	1.703	0.533
AC	GCCTGT	1965.56	2436	1.239	0.215
AC	GCGTGC	630.96	560	0.888	−0.119
AC	GCTTGT	1292.65	1142	0.883	−0.124
AC	GCATGT	1131.59	881	0.779	−0.250
AC	GCGTGT	531.45	322	0.606	−0.501
AC	GCTTGC	1534.70	894	0.583	−0.540
AC	GCATGC	1343.47	554	0.412	−0.886
AD	GCAGAT	2373.33	4215	1.776	0.574
AD	GCTGAT	2711.15	3887	1.434	0.360
AD	GCTGAC	3062.55	4374	1.428	0.356
AD	GCGGAC	1259.11	1625	1.291	0.255
AD	GCAGAC	2680.95	3395	1.266	0.236
AD	GCGGAT	1114.64	839	0.753	−0.284
AD	GCCGAC	4656.80	2726	0.585	−0.535
AD	GCCGAT	4122.47	920	0.223	−1.500
AE	GCAGAA	3517.48	5814	1.653	0.503
AE	GCAGAG	4703.98	7094	1.508	0.411
AE	GCGGAG	2209.23	3171	1.435	0.361
AE	GCTGAG	5373.53	7362	1.370	0.315
AE	GCTGAA	4018.14	5186	1.291	0.255
AE	GCCGAG	8170.80	5082	0.622	−0.475
AE	GCGGAA	1651.99	949	0.574	−0.554
AE	GCCGAA	6109.85	1097	0.180	−1.717
AF	GCCTTC	4447.90	7382	1.660	0.507
AF	GCATTT	2237.22	2332	1.042	0.041
AF	GCTTTT	2555.66	2580	1.010	0.009
AF	GCCTTT	3886.04	3842	0.989	−0.011
AF	GCTTTC	2925.16	2315	0.791	−0.234
AF	GCGTTC	1202.63	636	0.529	−0.637
AF	GCGTTT	1050.71	518	0.493	−0.707
AF	GCATTC	2560.68	1261	0.492	−0.708
AG	GCGGGC	1369.64	2638	1.926	0.655
AG	GCGGGG	986.17	1738	1.762	0.567
AG	GCTGGG	2398.67	3855	1.607	0.474
AG	GCTGGT	1590.73	2524	1.587	0.462
AG	GCTGGA	2457.02	3783	1.540	0.432
AG	GCAGGA	2150.87	3074	1.429	0.357
AG	GCAGGG	2099.79	2782	1.325	0.281
AG	GCAGGT	1392.52	1748	1.255	0.227
AG	GCTGGC	3331.38	3961	1.189	0.173
AG	GCAGGC	2916.28	3119	1.070	0.067
AG	GCGGGT	654.00	617	0.943	−0.058
AG	GCGGGA	1010.16	793	0.785	−0.242
AG	GCCGGG	3647.33	2240	0.614	−0.488
AG	GCCGGC	5065.58	2977	0.588	−0.532
AG	GCCGGT	2418.80	581	0.240	−1.426
AG	GCCGGA	3736.06	795	0.213	−1.547
AH	GCGCAC	748.29	983	1.314	0.273
AH	GCCCAC	2767.53	3465	1.252	0.225
AH	GCTCAT	1319.86	1471	1.115	0.108
AH	GCACAT	1155.40	1122	0.971	−0.029
AH	GCCCAT	2006.93	1827	0.910	−0.094
AH	GCTCAC	1820.07	1526	0.838	−0.176
AH	GCACAC	1593.29	1312	0.823	−0.194
AH	GCGCAT	542.64	248	0.457	−0.783
AI	GCCATC	3894.51	7798	2.002	0.694
AI	GCCATT	3079.73	3761	1.221	0.200
AI	GCAATA	815.43	924	1.133	0.125
AI	GCAATT	1773.02	1684	0.950	−0.052
AI	GCCATA	1416.41	1257	0.887	−0.119
AI	GCTATT	2025.39	1709	0.844	−0.170
AI	GCTATA	931.50	771	0.828	−0.189
AI	GCTATC	2561.23	1194	0.466	−0.763
AI	GCGATT	832.70	373	0.448	−0.803
AI	GCAATC	2242.09	984	0.439	−0.824
AI	GCGATA	382.97	149	0.389	−0.944
AI	GCGATC	1053.00	404	0.384	−0.958
AK	GCCAAG	5767.01	9818	1.702	0.532
AK	GCAAAA	2563.57	3011	1.175	0.161
AK	GCCAAA	4452.91	4794	1.077	0.074
AK	GCAAAG	3320.10	3044	0.917	−0.087
AK	GCTAAA	2928.46	2022	0.690	−0.370
AK	GCGAAG	1559.29	765	0.491	−0.712
AK	GCTAAG	3792.68	1725	0.455	−0.788
AK	GCGAAA	1203.98	409	0.340	−1.080
AL	GCGCTG	2369.16	4619	1.950	0.668
AL	GCGCTC	1140.05	1765	1.548	0.437
AL	GCTTTG	1873.51	2601	1.388	0.328
AL	GCCCTG	8762.30	11409	1.302	0.264
AL	GCCTTG	2848.79	3695	1.297	0.260
AL	GCTTTA	1115.24	1385	1.242	0.217
AL	GCCCTC	4216.45	4499	1.067	0.065
AL	GCTCTT	1912.07	2038	1.066	0.064
AL	GCATTA	976.28	986	1.010	0.010
AL	GCTCTA	1031.16	940	0.912	−0.093
AL	GCACTT	1673.82	1444	0.863	−0.148
AL	GCATTG	1640.07	1364	0.832	−0.184
AL	GCACTA	902.68	747	0.828	−0.189
AL	GCGCTA	423.94	342	0.807	−0.215
AL	GCCCTA	1567.95	1228	0.783	−0.244
AL	GCTCTG	5762.53	4505	0.782	−0.246
AL	GCCCTT	2907.42	2230	0.767	−0.265
AL	GCTCTC	2772.95	2036	0.734	−0.309
AL	GCCTTA	1695.80	1205	0.711	−0.342
AL	GCACTG	5044.51	3522	0.698	−0.359
AL	GCGTTG	770.26	476	0.618	−0.481
AL	GCGCTT	786.11	459	0.584	−0.538
AL	GCACTC	2427.43	1415	0.583	−0.540
AL	GCGTTA	458.51	169	0.369	−0.998
AM	GCCATG	4236.47	6521	1.539	0.431
AM	GCAATG	2438.96	1900	0.779	−0.250
AM	GCTATG	2786.11	1561	0.560	−0.579
AM	GCGATG	1145.46	625	0.546	−0.606
AN	GCCAAC	3190.28	5452	1.709	0.536
AN	GCAAAT	1667.60	2282	1.368	0.314
AN	GCCAAT	2896.62	3122	1.078	0.075
AN	GCAAAC	1836.66	1512	0.823	−0.195
AN	GCTAAT	1904.97	1356	0.712	−0.340
AN	GCTAAC	2098.09	925	0.441	−0.819
AN	GCGAAC	862.59	331	0.384	−0.958
AN	GCGAAT	783.19	260	0.332	−1.103
AP	GCGCCG	406.74	1172	2.881	1.058
AP	GCGCCC	1122.56	2271	2.023	0.705
AP	GCCCCG	1504.34	2335	1.552	0.440
AP	GCTCCA	2360.19	2463	1.044	0.043
AP	GCTCCT	2445.47	2548	1.042	0.041
AP	GCCCCC	4151.78	3957	0.953	−0.048
AP	GCACCT	2140.76	2028	0.947	−0.054
AP	GCCCCA	3588.82	3371	0.939	−0.063
AP	GCACCA	2066.10	1831	0.886	−0.121
AP	GCACCC	2390.20	2111	0.883	−0.124
AP	GCCCCT	3718.49	3269	0.879	−0.129
AP	GCTCCC	2730.42	2384	0.873	−0.136
AP	GCTCCG	989.33	773	0.781	−0.247
AP	GCGCCT	1005.41	778	0.774	−0.256
AP	GCACCG	866.06	571	0.659	−0.417
AP	GCGCCA	970.35	595	0.613	−0.489
AQ	GCCCAG	7143.67	9550	1.337	0.290
AQ	GCGCAG	1931.51	2101	1.088	0.084
AQ	GCACAA	1472.79	1416	0.961	−0.039
AQ	GCTCAA	1682.42	1522	0.905	−0.100
AQ	GCTCAG	4698.04	4141	0.881	−0.126
AQ	GCACAG	4112.65	3374	0.820	−0.198
AQ	GCCCAA	2558.23	1943	0.760	−0.275
AQ	GCGCAA	691.70	244	0.353	−1.042
AR	GCGCGC	580.17	1255	2.163	0.772
AR	GCGCGG	634.54	1175	1.852	0.616
AR	GCCCGG	2346.82	3946	1.681	0.520
AR	GCCCGC	2145.76	3135	1.461	0.379
AR	GCCAGG	2323.57	3242	1.395	0.333
AR	GCAAGA	1362.59	1559	1.144	0.135
AR	GCTCGA	836.64	943	1.127	0.120
AR	GCCCGA	1272.16	1418	1.115	0.109
AR	GCCCGT	918.67	935	1.018	0.018
AR	GCTCGT	604.17	595	0.985	−0.015
AR	GCCAGA	2366.81	2219	0.938	−0.064
AR	GCTCGG	1543.39	1295	0.839	−0.175
AR	GCGCGT	248.39	205	0.825	−0.192
AR	GCAAGG	1337.69	1089	0.814	−0.206
AR	GCGAGG	628.25	486	0.774	−0.257
AR	GCACGA	732.39	533	0.728	−0.318
AR	GCTCGC	1411.16	941	0.667	−0.405
AR	GCGCGA	343.97	226	0.657	−0.420
AR	GCACGT	528.89	338	0.639	−0.448
AR	GCACGG	1351.08	859	0.636	−0.453
AR	GCACGC	1235.33	619	0.501	−0.691
AR	GCTAGA	1556.53	714	0.459	−0.779
AR	GCGAGA	639.94	263	0.411	−0.889
AR	GCTAGG	1528.10	487	0.319	−1.144
AS	GCCTCG	963.41	1977	2.052	0.719
AS	GCGTCG	260.49	465	1.785	0.579
AS	GCCAGC	4127.58	6466	1.567	0.449
AS	GCCTCC	3643.21	5443	1.494	0.401
AS	GCTTCT	2084.25	2488	1.194	0.177
AS	GCCAGT	2604.12	3085	1.185	0.169
AS	GCATCT	1824.55	2154	1.181	0.166
AS	GCTTCA	1684.99	1932	1.147	0.137
AS	GCGTCC	985.05	1079	1.095	0.091
AS	GCATCA	1475.04	1531	1.038	0.037
AS	GCCTCT	3169.23	3235	1.021	0.021
AS	GCCTCA	2562.14	2514	0.981	−0.019
AS	GCTTCC	2395.96	2295	0.958	−0.043
AS	GCAAGT	1499.21	1307	0.872	−0.137
AS	GCTTCG	633.59	516	0.814	−0.205
AS	GCATCC	2097.42	1658	0.790	−0.235
AS	GCATCG	554.64	403	0.727	−0.319
AS	GCGTCT	856.90	521	0.608	−0.498
AS	GCGAGC	1116.02	595	0.533	−0.629
AS	GCGTCA	692.75	319	0.460	−0.775
AS	GCAAGC	2376.27	1080	0.454	−0.789
AS	GCTAGT	1712.60	737	0.430	−0.843
AS	GCGAGT	704.10	265	0.376	−0.977
AS	GCTAGC	2714.51	673	0.248	−1.395
AT	GCCACG	1262.40	2478	1.963	0.674
AT	GCCACC	3842.98	6598	1.717	0.541
AT	GCCACA	3111.04	4031	1.296	0.259
AT	GCCACT	2751.18	3205	1.165	0.153
AT	GCAACA	1791.05	1761	0.983	−0.017
AT	GCGACG	341.33	329	0.964	−0.037
AT	GCAACT	1583.87	1509	0.953	−0.048
AT	GCTACT	1809.31	1395	0.771	−0.260
AT	GCTACA	2045.98	1528	0.747	−0.292
AT	GCGACC	1039.07	601	0.578	−0.547
AT	GCAACC	2212.43	1259	0.569	−0.564
AT	GCTACC	2527.34	1364	0.540	−0.617
AT	GCAACG	726.77	384	0.528	−0.638
AT	GCTACG	830.22	363	0.437	−0.827
AT	GCGACT	743.87	308	0.414	−0.882
AT	GCGACA	841.17	347	0.413	−0.885
AV	GCTGTT	1736.99	3025	1.742	0.555
AV	GCTGTG	4399.56	7279	1.654	0.503
AV	GCTGTA	1127.89	1750	1.552	0.439
AV	GCTGTC	2223.90	3351	1.507	0.410
AV	GCAGTA	987.35	1401	1.419	0.350
AV	GCGGTG	1808.80	2487	1.375	0.318
AV	GCAGTT	1520.56	2087	1.373	0.317
AV	GCAGTG	3851.36	4349	1.129	0.122
AV	GCGGTC	914.32	883	0.966	−0.035
AV	GCAGTC	1946.80	1806	0.928	−0.075
AV	GCCGTG	6689.81	4322	0.646	−0.437
AV	GCGGTT	714.13	423	0.592	−0.524
AV	GCGGTA	463.71	270	0.582	−0.541
AV	GCCGTC	3381.59	1798	0.532	−0.632
AV	GCCGTT	2641.21	563	0.213	−1.546
AV	GCCGTA	1715.03	329	0.192	−1.651
AW	GCCTGG	2528.22	3848	1.522	0.420
AW	GCGTGG	683.58	558	0.816	−0.203
AW	GCTTGG	1662.69	1066	0.641	−0.445
AW	GCATGG	1455.51	858	0.589	−0.529
AY	GCCTAC	2643.77	4073	1.541	0.432
AY	GCCTAT	2148.26	2457	1.144	0.134
AY	GCTTAT	1412.81	1478	1.046	0.045
AY	GCATAT	1236.77	1244	1.006	0.006
AY	GCTTAC	1738.68	1139	0.655	−0.423
AY	GCGTAC	714.83	429	0.600	−0.511
AY	GCATAC	1522.04	868	0.570	−0.562
AY	GCGTAT	580.85	310	0.534	−0.628
CA	TGTGCT	1164.04	2021	1.736	0.552
CA	TGTGCC	1769.99	2992	1.690	0.525
CA	TGTGCA	1019.00	1708	1.676	0.517
CA	TGTGCG	478.57	477	0.997	−0.003
CA	TGCGCG	568.18	502	0.884	−0.124
CA	TGCGCC	2101.42	1313	0.625	−0.470
CA	TGCGCT	1382.00	368	0.266	−1.323
CA	TGCGCA	1209.80	312	0.258	−1.355
CC	TGCTGC	1534.17	2610	1.701	0.531
CC	TGCTGT	1292.21	1571	1.216	0.195
CC	TGTTGT	1088.41	529	0.486	−0.721
CC	TGTTGC	1292.21	497	0.385	−0.956
CD	TGTGAC	1920.20	3470	1.807	0.592
CD	TGTGAT	1699.87	2853	1.678	0.518
CD	TGCGAC	2279.75	1134	0.497	−0.698
CD	TGCGAT	2018.17	461	0.228	−1.477
CE	TGTGAA	1901.69	3636	1.912	0.648
CE	TGTGAG	2543.16	3935	1.547	0.437
CE	TGCGAG	3019.37	1709	0.566	−0.569
CE	TGCGAA	2257.78	442	0.196	−1.631
CF	TGCTTC	1891.74	2684	1.419	0.350
CF	TGCTTT	1652.78	1685	1.019	0.019
CF	TGTTTT	1392.11	1096	0.787	−0.239
CF	TGTTTC	1593.38	1065	0.668	−0.403
CG	TGTGGG	1594.78	3240	2.032	0.709
CG	TGTGGA	1633.57	2846	1.742	0.555
CG	TGTGGT	1057.61	1627	1.538	0.431
CG	TGTGGC	2214.90	3133	1.415	0.347
CG	TGCGGG	1893.40	1137	0.601	−0.510
CG	TGCGGC	2629.63	1461	0.556	−0.588
CG	TGCGGT	1255.64	344	0.274	−1.295
CG	TGCGGA	1939.46	431	0.222	−1.504
CH	TGCCAC	1618.50	2144	1.325	0.281
CH	TGCCAT	1173.68	1253	1.068	0.065
CH	TGTCAT	988.58	831	0.841	−0.174
CH	TGTCAC	1363.24	916	0.672	−0.398
CI	TGCATC	1821.04	2813	1.545	0.435
CI	TGCATT	1440.05	1579	1.096	0.092
CI	TGCATA	662.30	576	0.870	−0.140
CI	TGTATA	557.84	474	0.850	−0.163
CI	TGTATT	1212.94	927	0.764	−0.269
CI	TGTATC	1533.83	859	0.560	−0.580
CK	TGCAAG	2777.53	3348	1.205	0.187
CK	TGCAAA	2144.62	2441	1.138	0.129
CK	TGTAAA	1806.38	1770	0.980	−0.020
CK	TGTAAG	2339.47	1509	0.645	−0.438
CL	TGCCTC	1722.14	2468	1.433	0.360
CL	TGCCTG	3578.83	4525	1.264	0.235
CL	TGTTTA	583.38	704	1.207	0.188
CL	TGCCTT	1187.49	1384	1.165	0.153
CL	TGTTTG	980.04	1079	1.101	0.096
CL	TGCTTG	1163.55	1179	1.013	0.013
CL	TGTCTT	1000.21	940	0.940	−0.062
CL	TGCCTA	640.41	585	0.913	−0.090
CL	TGTCTA	539.40	481	0.892	−0.115
CL	TGCTTA	692.62	565	0.816	−0.204
CL	TGTCTC	1450.53	1010	0.696	−0.362
CL	TGTCTG	3014.39	1633	0.542	−0.613
CM	TGCATG	1518.22	1979	1.304	0.265
CM	TGTATG	1278.78	818	0.640	−0.447
CN	TGCAAC	1825.04	2351	1.288	0.253
CN	TGCAAT	1657.05	1636	0.987	−0.013
CN	TGTAAT	1395.71	1349	0.967	−0.034
CN	TGTAAC	1537.20	1079	0.702	−0.354
CP	TGCCCG	687.28	978	1.423	0.353
CP	TGCCCC	1896.80	2279	1.201	0.184
CP	TGCCCA	1639.61	1728	1.054	0.053
CP	TGCCCT	1698.85	1690	0.995	−0.005
CP	TGTCCT	1430.91	1333	0.932	−0.071
CP	TGTCCA	1381.01	1263	0.915	−0.089
CP	TGTCCC	1597.65	1369	0.857	−0.154
CP	TGTCCG	578.88	271	0.468	−0.759
CQ	TGCCAG	3338.89	4321	1.294	0.258
CQ	TGCCAA	1195.69	1319	1.103	0.098
CQ	TGTCAA	1007.11	905	0.899	−0.107
CQ	TGTCAG	2812.30	1809	0.643	−0.441
CR	TGCCGC	1031.52	1860	1.803	0.590
CR	TGCCGG	1128.18	1543	1.368	0.313
CR	TGCAGG	1117.00	1450	1.298	0.261
CR	TGCCGT	441.63	541	1.225	0.203
CR	TGCCGA	611.56	742	1.213	0.193
CR	TGCAGA	1137.78	1252	1.100	0.096
CR	TGTCGA	515.11	458	0.889	−0.118
CR	TGTCGT	371.98	308	0.828	−0.189
CR	TGTAGA	958.34	570	0.595	−0.520
CR	TGTCGC	868.83	497	0.572	−0.559
CR	TGTCGG	950.24	463	0.487	−0.719
CR	TGTAGG	940.83	389	0.413	−0.883
CS	TGCAGC	1990.73	3150	1.582	0.459
CS	TGCTCC	1757.12	2397	1.364	0.311
CS	TGCAGT	1255.97	1701	1.354	0.303
CS	TGCTCG	464.65	571	1.229	0.206
CS	TGTTCT	1287.45	1184	0.920	−0.084
CS	TGCTCT	1528.52	1393	0.911	−0.093
CS	TGTTCA	1040.83	932	0.895	−0.110
CS	TGCTCA	1235.72	1079	0.873	−0.136
CS	TGTTCC	1479.99	1102	0.745	−0.295
CS	TGTAGT	1057.88	699	0.661	−0.414
CS	TGTTCG	391.37	192	0.491	−0.712
CS	TGTAGC	1676.76	767	0.457	−0.782
CT	TGCACG	535.88	829	1.547	0.436
CT	TGCACC	1631.31	2321	1.423	0.353
CT	TGCACA	1320.60	1508	1.142	0.133
CT	TGCACT	1167.85	1185	1.015	0.015
CT	TGTACT	983.66	802	0.815	−0.204
CT	TGTACA	1112.32	830	0.746	−0.293
CT	TGTACC	1374.02	942	0.686	−0.377
CT	TGTACG	451.36	160	0.354	−1.037
CV	TGTGTC	1064.94	1821	1.710	0.536
CV	TGTGTT	831.78	1383	1.663	0.508
CV	TGTGTA	540.10	866	1.603	0.472
CV	TGTGTG	2106.78	3241	1.538	0.431
CV	TGCGTG	2501.27	1537	0.614	−0.487
CV	TGCGTC	1264.35	734	0.581	−0.544
CV	TGCGTT	987.53	219	0.222	−1.506
CV	TGCGTA	641.24	137	0.214	−1.543
CW	TGCTGG	1275.05	1842	1.445	0.368
CW	TGTTGG	1073.95	507	0.472	−0.751
CY	TGCTAC	1379.34	1995	1.446	0.369
CY	TGCTAT	1120.82	1170	1.044	0.043
CY	TGTTAT	944.05	653	0.692	−0.369
CY	TGTTAC	1161.80	788	0.678	−0.388
DA	GATGCT	2675.13	5292	1.978	0.682
DA	GATGCA	2341.80	3898	1.665	0.510
DA	GATGCC	4067.71	5983	1.471	0.386
DA	GACGCG	1242.39	1116	0.898	−0.107
DA	GATGCG	1099.83	972	0.884	−0.124
DA	GACGCC	4594.94	2668	0.581	−0.544
DA	GACGCA	2645.34	852	0.322	−1.133
DA	GACGCT	3021.87	908	0.300	−1.202
DC	GACTGC	2386.86	3465	1.452	0.373
DC	GACTGT	2010.41	2804	1.395	0.333
DC	GATTGT	1779.74	1163	0.653	−0.425
DC	GATTGC	2112.99	858	0.406	−0.901
DD	GATGAT	4271.42	7846	1.837	0.608
DD	GATGAC	4825.06	7181	1.488	0.398
DD	GACGAC	5450.46	2965	0.544	−0.609
DD	GACGAT	4825.06	1380	0.286	−1.252
DE	GATGAA	5114.33	10045	1.964	0.675
DE	GATGAG	6839.48	9573	1.400	0.336
DE	GACGAG	7725.97	4498	0.582	−0.541
DE	GACGAA	5777.22	1341	0.232	−1.461
DF	GACTTC	4696.28	6094	1.298	0.261
DF	GACTTT	4103.05	4250	1.036	0.035
DF	GATTTT	3632.26	3485	0.959	−0.041
DF	GATTTC	4157.42	2760	0.664	−0.410
DG	GATGGT	1910.36	3443	1.802	0.589
DG	GATGGA	2950.72	5133	1.740	0.554
DG	GATGGG	2880.65	4437	1.540	0.432
DG	GATGGC	4000.77	5419	1.354	0.303
DG	GACGGC	4519.33	2987	0.661	−0.414
DG	GACGGG	3254.02	1979	0.608	−0.497
DG	GACGGT	2157.97	723	0.335	−1.094
DG	GACGGA	3333.18	886	0.266	−1.325
DH	GACCAC	2653.74	3480	1.311	0.271
DH	GACCAT	1924.41	2014	1.047	0.046
DH	GATCAT	1703.60	1623	0.953	−0.048
DH	GATCAC	2349.25	1514	0.644	−0.439
DI	GACATC	4715.94	6532	1.385	0.326
DI	GACATT	3729.31	4087	1.096	0.092
DI	GATATT	3301.40	3271	0.991	−0.009
DI	GATATA	1518.36	1495	0.985	−0.016
DI	GACATA	1715.16	1565	0.912	−0.092
DI	GATATC	4174.83	2205	0.528	−0.638
DK	GACAAG	5562.52	7324	1.317	0.275
DK	GACAAA	4295.02	4794	1.116	0.110
DK	GATAAA	3802.20	3855	1.014	0.014
DK	GATAAG	4924.27	2611	0.530	−0.634
DL	GACCTC	3785.97	5029	1.328	0.284
DL	GACTTG	2557.95	3396	1.328	0.283
DL	GATTTA	1347.95	1740	1.291	0.255
DL	GACCTG	7867.71	9796	1.245	0.219
DL	GATTTG	2264.44	2687	1.187	0.171
DL	GACCTT	2610.58	2774	1.063	0.061
DL	GATCTT	2311.04	2416	1.045	0.044
DL	GACCTA	1407.87	1416	1.006	0.006
DL	GACTTA	1522.66	1403	0.921	−0.082
DL	GATCTA	1246.33	1020	0.818	−0.200
DL	GATCTC	3351.56	2214	0.661	−0.415
DL	GATCTG	6964.95	3348	0.481	−0.733
DM	GACATG	4089.63	5411	1.323	0.280
DM	GATATG	3620.37	2299	0.635	−0.454
DN	GACAAC	3511.00	4849	1.381	0.323
DN	GACAAT	3187.82	3349	1.051	0.049
DN	GATAAT	2822.05	2549	0.903	−0.102
DN	GATAAC	3108.14	1882	0.606	−0.502
DP	GACCCC	3732.11	5119	1.372	0.316
DP	GACCCG	1352.28	1692	1.251	0.224
DP	GACCCT	3342.62	3700	1.107	0.102
DP	GATCCT	2959.08	3111	1.051	0.050
DP	GACCCA	3226.05	3205	0.993	−0.007
DP	GATCCA	2855.89	2349	0.823	−0.195
DP	GATCCC	3303.88	2338	0.708	−0.346
DP	GATCCG	1197.11	455	0.380	−0.967
DQ	GACCAG	5250.37	6524	1.243	0.217
DQ	GACCAA	1880.22	2169	1.154	0.143
DQ	GATCAA	1664.48	1808	1.086	0.083
DQ	GATCAG	4647.93	2942	0.633	−0.457
DR	GACCGC	1807.77	2634	1.457	0.376
DR	GACAGA	1994.00	2869	1.439	0.364
DR	GACAGG	1957.57	2730	1.395	0.333
DR	GACCGT	773.97	1029	1.330	0.285
DR	GACCGG	1977.16	2568	1.299	0.261
DR	GACCGA	1071.78	1292	1.205	0.187
DR	GATCGA	948.80	923	0.973	−0.028
DR	GATCGT	685.16	626	0.914	−0.090
DR	GATAGA	1765.20	1123	0.636	−0.452
DR	GATCGG	1750.30	859	0.491	−0.712
DR	GATCGC	1600.34	754	0.471	−0.753
DR	GATAGG	1732.96	658	0.380	−0.968
DS	GACTCG	918.57	1527	1.662	0.508
DS	GACAGC	3935.48	6143	1.561	0.445
DS	GACAGT	2482.92	3657	1.473	0.387
DS	GATTCT	2675.01	2968	1.110	0.104
DS	GACTCC	3473.65	3800	1.094	0.090
DS	GATTCA	2162.59	2129	0.984	−0.016
DS	GACTCA	2442.89	2382	0.975	−0.025
DS	GACTCT	3021.73	2910	0.963	−0.038
DS	GATTCC	3075.07	2186	0.711	−0.341
DS	GATAGT	2198.02	1355	0.616	−0.484
DS	GATTCG	813.17	414	0.509	−0.675
DS	GATAGC	3483.91	1212	0.348	−1.056
DT	GACACG	1110.58	1842	1.659	0.506
DT	GACACC	3380.79	4666	1.380	0.322
DT	GACACA	2736.88	3538	1.293	0.257
DT	GACACT	2420.30	2688	1.111	0.105
DT	GATACT	2142.59	1731	0.808	−0.213
DT	GATACA	2422.85	1788	0.738	−0.304
DT	GATACC	2992.87	1586	0.530	−0.635
DT	GATACG	983.15	351	0.357	−1.030
DV	GATGTT	1957.96	3699	1.889	0.636
DV	GATGTA	1271.37	2214	1.741	0.555
DV	GATGTC	2506.81	3869	1.543	0.434
DV	GATGTG	4959.23	6668	1.345	0.296
DV	GACGTG	5602.02	3616	0.645	−0.438
DV	GACGTC	2831.73	1654	0.584	−0.538
DV	GACGTT	2211.73	672	0.304	−1.191
DV	GACGTA	1436.16	385	0.268	−1.316
DW	GACTGG	2619.27	3853	1.471	0.386
DW	GATTGG	2318.73	1085	0.468	−0.759
DY	GACTAC	3307.71	3930	1.188	0.172
DY	GATTAT	2379.36	2608	1.096	0.092
DY	GACTAT	2687.76	2853	1.061	0.060
DY	GATTAC	2928.18	1912	0.653	−0.426
EA	GAGGCG	2437.29	3179	1.304	0.266
EA	GAAGCA	3880.59	4844	1.248	0.222
EA	GAAGCT	4432.94	5143	1.160	0.149
EA	GAGGCC	9014.27	9805	1.088	0.084
EA	GAGGCT	5928.25	5314	0.896	−0.109
EA	GAGGCA	5189.57	4530	0.873	−0.136
EA	GAAGCC	6740.57	5649	0.838	−0.177
EA	GAAGCG	1822.52	982	0.539	−0.618
EC	GAATGT	2182.58	3541	1.622	0.484
EC	GAGTGT	2918.80	2792	0.957	−0.044
EC	GAGTGC	3465.35	2987	0.862	−0.149
EC	GAATGC	2591.27	1838	0.709	−0.343
ED	GAAGAT	6605.82	9691	1.467	0.383
ED	GAGGAC	9979.09	9684	0.970	−0.030
ED	GAAGAC	7462.02	6820	0.914	−0.090
ED	GAGGAT	8834.07	6686	0.757	−0.279
EE	GAAGAA	10747.11	14461	1.346	0.297
EE	GAGGAG	19220.31	21731	1.131	0.123
EE	GAAGAG	14372.29	11875	0.826	−0.191
EE	GAGGAA	14372.29	10645	0.741	−0.300
EF	GAATTT	3136.91	4237	1.351	0.301
EF	GAGTTC	4801.58	4739	0.987	−0.013
EF	GAGTTT	4195.05	4095	0.976	−0.024
EF	GAATTC	3590.46	2653	0.739	−0.303
EG	GAAGGA	3358.73	5032	1.498	0.404
EG	GAAGGT	2174.51	2839	1.306	0.267
EG	GAAGGG	3278.97	3559	1.085	0.082
EG	GAGGGC	6090.10	6505	1.068	0.066
EG	GAAGGC	4553.97	4340	0.953	−0.048
EG	GAGGGG	4385.02	3795	0.865	−0.145
EG	GAGGGT	2908.01	2378	0.818	−0.201
EG	GAGGGA	4491.69	2793	0.622	−0.475
EH	GAACAT	2017.28	2539	1.259	0.230
EH	GAGCAC	3720.16	4190	1.126	0.119
EH	GAGCAT	2697.74	2448	0.907	−0.097
EH	GAACAC	2781.81	2040	0.733	−0.310
EI	GAAATA	1687.78	3007	1.782	0.578
EI	GAAATT	3669.78	4788	1.305	0.266
EI	GAGATC	6206.03	6191	0.998	−0.002
EI	GAGATT	4907.66	3978	0.811	−0.210
EI	GAGATA	2257.09	1785	0.791	−0.235
EI	GAAATC	4640.66	3620	0.780	−0.248
EK	GAGAAG	12729.57	15133	1.189	0.173
EK	GAAAAA	7349.75	7522	1.023	0.023
EK	GAGAAA	9828.94	9127	0.929	−0.074
EK	GAAAAG	9518.74	7645	0.803	−0.219
EL	GAGCTG	10945.64	15625	1.428	0.356
EL	GAATTA	1584.03	2256	1.424	0.354
EL	GAACTA	1464.61	1830	1.249	0.223
EL	GAACTT	2715.79	3371	1.241	0.216
EL	GAGCTC	5267.08	5877	1.116	0.110
EL	GAGCTA	1958.64	2049	1.046	0.045
EL	GAATTG	2661.03	2335	0.877	−0.131
EL	GAGCTT	3631.87	3084	0.849	−0.164
EL	GAGTTG	3558.64	2719	0.764	−0.269
EL	GAACTC	3938.54	2632	0.668	−0.403
EL	GAGTTA	2118.35	1357	0.641	−0.445
EL	GAACTG	8184.78	4894	0.598	−0.514
EM	GAAATG	4983.92	5010	1.005	0.005
EM	GAGATG	6665.08	6639	0.996	−0.004
EN	GAAAAT	4791.73	6977	1.456	0.376
EN	GAGAAC	7057.70	6756	0.957	−0.044
EN	GAAAAC	5277.51	4930	0.934	−0.068
EN	GAGAAT	6408.07	4872	0.760	−0.274
EP	GAGCCG	1650.94	2438	1.477	0.390
EP	GAGCCC	4556.38	6270	1.376	0.319
EP	GAGCCT	4080.86	4236	1.038	0.037
EP	GAGCCA	3938.55	4067	1.033	0.032
EP	GAACCA	2945.12	2684	0.911	−0.093
EP	GAACCT	3051.53	2547	0.835	−0.181
EP	GAACCC	3407.10	2106	0.618	−0.481
EP	GAACCG	1234.52	517	0.419	−0.870
EQ	GAACAA	2579.50	3396	1.317	0.275
EQ	GAGCAG	9632.80	11185	1.161	0.149
EQ	GAGCAA	3449.61	3185	0.923	−0.080
EQ	GAACAG	7203.08	5099	0.708	−0.345
ER	GAAAGA	2650.27	3769	1.422	0.352
ER	GAGAGG	3479.50	4315	1.240	0.215
ER	GAGCGG	3514.32	4356	1.240	0.215
ER	GAGCGC	3213.23	3682	1.146	0.136
ER	GAAAGG	2601.85	2679	1.030	0.029
ER	GAGAGA	3544.25	3633	1.025	0.025
ER	GAGCGT	1375.70	1286	0.935	−0.067
ER	GAACGT	1028.70	894	0.869	−0.140
ER	GAACGA	1424.52	1188	0.834	−0.182
ER	GAGCGA	1905.04	1562	0.820	−0.199
ER	GAACGG	2627.88	1333	0.507	−0.679
ER	GAACGC	2402.74	1071	0.446	−0.808
ES	GAAAGT	2081.93	3138	1.507	0.410
ES	GAGAGC	4413.03	5786	1.311	0.271
ES	GAGAGT	2784.21	3237	1.163	0.151
ES	GAGTCG	1030.03	1174	1.140	0.131
ES	GAATCT	2533.73	2812	1.110	0.104
ES	GAATCA	2048.37	2131	1.040	0.040
ES	GAAAGC	3299.91	2880	0.873	−0.136
ES	GAGTCC	3895.16	3392	0.871	−0.138
ES	GAGTCT	3388.40	2799	0.826	−0.191
ES	GAGTCA	2739.33	2198	0.802	−0.220
ES	GAATCC	2912.67	1943	0.667	−0.405
ES	GAATCG	770.22	407	0.528	−0.638
ET	GAGACG	1658.42	2190	1.321	0.278
ET	GAAACA	3056.09	3851	1.260	0.231
ET	GAAACT	2702.59	3224	1.193	0.176
ET	GAGACC	5048.51	5514	1.092	0.088
ET	GAGACA	4086.97	3619	0.885	−0.122
ET	GAGACT	3614.21	3028	0.838	−0.177
ET	GAAACC	3775.11	2950	0.781	−0.247
ET	GAAACG	1240.11	806	0.650	−0.431
EV	GAAGTA	1580.16	2675	1.693	0.526
EV	GAAGTT	2433.50	3724	1.530	0.425
EV	GAGGTG	8242.83	9074	1.101	0.096
EV	GAAGTC	3115.66	2860	0.918	−0.086
EV	GAGGTC	4166.62	3741	0.898	−0.108
EV	GAAGTG	6163.71	5122	0.831	−0.185
EV	GAGGTT	3254.36	2359	0.725	−0.322
EV	GAGGTA	2113.17	1515	0.717	−0.333
EW	GAGTGG	3085.08	3238	1.050	0.048
EW	GAATGG	2306.92	2154	0.934	−0.069
EY	GAATAT	2307.55	3428	1.486	0.396
EY	GAGTAC	3797.72	3796	1.000	0.000
EY	GAGTAT	3085.93	2596	0.841	−0.173
EY	GAATAC	2839.80	2211	0.779	−0.250
FA	TTTGCA	1643.98	3299	2.007	0.696
FA	TTTGCT	1877.98	3746	1.995	0.690
FA	TTTGCC	2855.59	4348	1.523	0.420
FA	TTTGCG	772.10	622	0.806	−0.216
FA	TTCGCG	883.73	598	0.677	−0.391
FA	TTCGCC	3268.46	1802	0.551	−0.595
FA	TTCGCT	2149.50	516	0.240	−1.427
FA	TTCGCA	1881.67	402	0.214	−1.543
FC	TTCTGC	2058.60	3045	1.479	0.391
FC	TTCTGT	1733.93	2055	1.185	0.170
FC	TTTTGT	1514.90	1159	0.765	−0.268
FC	TTTTGC	1798.56	847	0.471	−0.753
FD	TTTGAT	2786.65	5380	1.931	0.658
FD	TTTGAC	3147.84	4737	1.505	0.409
FD	TTCGAC	3602.96	1746	0.485	−0.724
FD	TTCGAT	3189.55	864	0.271	−1.306
FE	TTTGAA	3016.02	6247	2.071	0.728
FE	TTTGAG	4033.37	6066	1.504	0.408
FE	TTCGAG	4616.53	2165	0.469	−0.757
FE	TTCGAA	3452.08	640	0.185	−1.685
FF	TTCTTC	3429.53	5168	1.507	0.410
FF	TTCTTT	2996.32	2989	0.998	−0.002
FF	TTTTTT	2617.83	1937	0.740	−0.301
FF	TTTTTC	2996.32	1946	0.649	−0.432
FG	TTTGGA	2068.21	4271	2.065	0.725
FG	TTTGGT	1339.00	2552	1.906	0.645
FG	TTTGGG	2019.09	3449	1.708	0.535
FG	TTTGGC	2804.20	3462	1.235	0.211
FG	TTCGGG	2311.02	1292	0.559	−0.581
FG	TTCGGC	3209.64	1648	0.513	−0.667
FG	TTCGGT	1532.60	419	0.273	−1.297
FG	TTCGGA	2367.24	558	0.236	−1.445
FH	TTCCAC	2463.48	3200	1.299	0.262
FH	TTTCAT	1560.78	1697	1.087	0.084
FH	TTCCAT	1786.44	1866	1.045	0.044
FH	TTTCAC	2152.30	1200	0.558	−0.584
FI	TTCATC	3454.46	5156	1.493	0.400
FI	TTCATT	2731.75	2953	1.081	0.078
FI	TTTATT	2386.67	2296	0.962	−0.039
FI	TTTATA	1097.66	950	0.865	−0.144
FI	TTCATA	1256.36	1035	0.824	−0.194
FI	TTTATC	3018.10	1555	0.515	−0.663
FK	TTCAAG	4090.45	5137	1.256	0.228
FK	TTCAAA	3158.38	3245	1.027	0.027
FK	TTTAAA	2759.42	2762	1.001	0.001
FK	TTTAAG	3573.75	2438	0.682	−0.382
FL	TTCCTC	3228.53	4426	1.371	0.315
FL	TTCCTG	6709.28	8734	1.302	0.264
FL	TTTTTA	1134.45	1334	1.176	0.162
FL	TTTCTT	1945.00	2267	1.166	0.153
FL	TTCCTA	1200.58	1280	1.066	0.064
FL	TTTCTA	1048.92	1087	1.036	0.036
FL	TTCTTG	2181.32	2239	1.026	0.026
FL	TTCCTT	2226.21	2150	0.966	−0.035
FL	TTTTTG	1905.78	1799	0.944	−0.058
FL	TTCTTA	1298.47	1144	0.881	−0.127
FL	TTTCTC	2820.70	1904	0.675	−0.393
FL	TTTCTG	5861.77	3197	0.545	−0.606
FM	TTCATG	2804.11	3662	1.306	0.267
FM	TTTATG	2449.89	1592	0.650	−0.431
FN	TTCAAC	2855.47	3919	1.372	0.317
FN	TTTAAT	2265.13	2185	0.965	−0.036
FN	TTCAAT	2592.63	2456	0.947	−0.054
FN	TTTAAC	2494.77	1648	0.661	−0.415
FP	TTCCCG	961.40	1205	1.253	0.226
FP	TTTCCT	2076.25	2539	1.223	0.201
FP	TTCCCC	2653.35	3099	1.168	0.155
FP	TTTCCA	2003.85	2141	1.068	0.066
FP	TTCCCA	2293.57	2310	1.007	0.007
FP	TTCCCT	2376.44	2379	1.001	0.001
FP	TTTCCC	2318.18	1529	0.660	−0.416
FP	TTTCCG	839.96	321	0.382	−0.962
FQ	TTCCAG	5468.69	7069	1.293	0.257
FQ	TTTCAA	1711.02	1803	1.054	0.052
FQ	TTCCAA	1958.40	1980	1.011	0.011
FQ	TTTCAG	4777.89	3064	0.641	−0.444
FR	TTCCGC	1531.47	2588	1.690	0.525
FR	TTCCGA	907.97	1410	1.553	0.440
FR	TTCCGG	1674.97	2451	1.463	0.381
FR	TTCCGT	655.68	893	1.362	0.309
FR	TTCAGA	1689.24	1852	1.096	0.092
FR	TTCAGG	1658.38	1810	1.091	0.087
FR	TTTCGA	793.28	850	1.072	0.069
FR	TTTCGT	572.85	490	0.855	−0.156
FR	TTTAGA	1475.86	947	0.642	−0.444
FR	TTTAGG	1448.90	691	0.477	−0.740
FR	TTTCGG	1463.39	688	0.470	−0.755
FR	TTTCGC	1338.02	540	0.404	−0.907
FS	TTCTCC	2990.83	4507	1.507	0.410
FS	TTCAGC	3388.47	4577	1.351	0.301
FS	TTCAGT	2137.80	2692	1.259	0.231
FS	TTCTCG	790.89	910	1.151	0.140
FS	TTTTCT	2273.08	2536	1.116	0.109
FS	TTCTCT	2601.73	2741	1.054	0.052
FS	TTTTCA	1837.65	1903	1.036	0.035
FS	TTCTCA	2103.34	1997	0.949	−0.052
FS	TTTTCC	2613.03	1872	0.716	−0.334
FS	TTTAGT	1867.76	1201	0.643	−0.442
FS	TTTTCG	690.99	258	0.373	−0.985
FS	TTTAGC	2960.44	1062	0.359	−1.025
FT	TTCACC	2909.29	4513	1.551	0.439
FT	TTCACG	955.69	1315	1.376	0.319
FT	TTCACT	2082.75	2494	1.197	0.180
FT	TTCACA	2355.18	2372	1.007	0.007
FT	TTTACT	1819.66	1622	0.891	−0.115
FT	TTTACA	2057.68	1485	0.722	−0.326
FT	TTTACC	2541.79	1495	0.588	−0.531
FT	TTTACG	834.97	261	0.313	−1.163
FV	TTTGTA	912.19	1711	1.876	0.629
FV	TTTGTT	1404.80	2620	1.865	0.623
FV	TTTGTC	1798.60	2635	1.465	0.382
FV	TTTGTG	3558.17	5206	1.463	0.381
FV	TTCGTG	4072.62	2589	0.636	−0.453
FV	TTCGTC	2058.64	1086	0.528	−0.640
FV	TTCGTT	1607.91	386	0.240	−1.427
FV	TTCGTA	1044.07	224	0.215	−1.539
FW	TTCTGG	2126.30	2834	1.333	0.287
FW	TTTTGG	1857.70	1150	0.619	−0.480
FY	TTCTAC	2720.70	3710	1.364	0.310
FY	TTTTAT	1931.51	2003	1.037	0.036
FY	TTCTAT	2210.77	2145	0.970	−0.030
FY	TTTTAC	2377.02	1382	0.581	−0.542
GA	GGTGCT	1531.20	2505	1.636	0.492
GA	GGGGCG	949.27	1433	1.510	0.412
GA	GGGGCC	3510.85	5061	1.442	0.366
GA	GGTGCC	2328.29	3109	1.335	0.289
GA	GGAGCA	2070.38	2678	1.293	0.257
GA	GGTGCA	1340.41	1715	1.279	0.246
GA	GGCGCG	1318.38	1659	1.258	0.230
GA	GGAGCT	2365.08	2975	1.258	0.229
GA	GGGGCT	2308.91	2850	1.234	0.211
GA	GGAGCC	3596.25	3845	1.069	0.067
GA	GGGGCA	2021.22	2074	1.026	0.026
GA	GGTGCG	629.52	501	0.796	−0.228
GA	GGAGCG	972.36	712	0.732	−0.312
GA	GGCGCC	4876.02	3121	0.640	−0.446
GA	GGCGCT	3206.72	906	0.283	−1.264
GA	GGCGCA	2807.15	688	0.245	−1.406
GC	GGCTGC	1888.96	4102	2.172	0.775
GC	GGCTGT	1591.04	2360	1.483	0.394
GC	GGTTGT	759.72	658	0.866	−0.144
GC	GGATGT	1173.45	793	0.676	−0.392
GC	GGTTGC	901.97	523	0.580	−0.545
GC	GGATGC	1393.18	655	0.470	−0.755
GC	GGGTGC	1360.09	628	0.462	−0.773
GC	GGGTGT	1145.59	495	0.432	−0.839
GD	GGGGAC	3126.50	4967	1.589	0.463
GD	GGTGAT	1835.49	2621	1.428	0.356
GD	GGTGAC	2073.40	2960	1.428	0.356
GD	GGAGAT	2835.09	3829	1.351	0.301
GD	GGAGAC	3202.56	4240	1.324	0.281
GD	GGGGAT	2767.76	2575	0.930	−0.072
GD	GGCGAC	4342.22	1955	0.450	−0.798
GD	GGCGAT	3843.98	880	0.229	−1.474
GE	GGAGAA	3433.99	5903	1.719	0.542
GE	GGGGAG	4483.27	6552	1.461	0.379
GE	GGTGAA	2223.23	3248	1.461	0.379
GE	GGAGAG	4592.33	5961	1.298	0.261
GE	GGTGAG	2973.17	2988	1.005	0.005
GE	GGGGAA	3352.44	3041	0.907	−0.098
GE	GGCGAG	6226.56	3530	0.567	−0.568
GE	GGCGAA	4656.01	718	0.154	−1.869
GF	GGCTTC	3466.22	6121	1.766	0.569
GF	GGATTT	2233.54	2666	1.194	0.177
GF	GGTTTT	1446.04	1665	1.151	0.141
GF	GGCTTT	3028.37	3201	1.057	0.055
GF	GGTTTC	1655.11	1548	0.935	−0.067
GF	GGATTC	2556.47	1534	0.600	−0.511
GF	GGGTTT	2180.50	1244	0.571	−0.561
GF	GGGTTC	2495.76	1083	0.434	−0.835
GG	GGTGGT	1061.28	2286	2.154	0.767
GG	GGTGGC	2222.59	3657	1.645	0.498
GG	GGTGGA	1639.25	2618	1.597	0.468
GG	GGAGGA	2531.97	3609	1.425	0.354
GG	GGTGGG	1600.32	2267	1.417	0.348
GG	GGGGGC	3351.47	4673	1.394	0.332
GG	GGAGGT	1639.25	2152	1.313	0.272
GG	GGAGGC	3433.00	3776	1.100	0.095
GG	GGCGGC	4654.67	4787	1.028	0.028
GG	GGGGGT	1600.32	1543	0.964	−0.036
GG	GGAGGG	2471.84	2351	0.951	−0.050
GG	GGGGGA	2471.84	1517	0.614	−0.488
GG	GGCGGG	3351.47	2001	0.597	−0.516
GG	GGGGGG	2413.14	1080	0.448	−0.804
GG	GGCGGT	2222.59	936	0.421	−0.865
GG	GGCGGA	3433.00	845	0.246	−1.402
GH	GGCCAC	2540.15	3679	1.448	0.370
GH	GGTCAT	879.57	1022	1.162	0.150
GH	GGACAT	1358.57	1438	1.058	0.057
GH	GGCCAT	1842.04	1679	0.911	−0.093
GH	GGGCAC	1828.97	1629	0.891	−0.116
GH	GGTCAC	1212.92	1008	0.831	−0.185
GH	GGACAC	1873.46	1479	0.789	−0.236
GH	GGGCAT	1326.31	928	0.700	−0.357
GI	GGCATC	3372.48	5474	1.623	0.484
GI	GGAATA	904.63	1338	1.479	0.391
GI	GGAATT	1966.96	2560	1.302	0.264
GI	GGCATT	2666.92	2670	1.001	0.001
GI	GGTATT	1273.45	1052	0.826	−0.191
GI	GGGATC	2428.27	1958	0.806	−0.215
GI	GGTATA	585.67	461	0.787	−0.239
GI	GGAATC	2487.34	1910	0.768	−0.264
GI	GGGATA	883.14	666	0.754	−0.282
GI	GGGATT	1920.24	1421	0.740	−0.301
GI	GGCATA	1226.55	885	0.722	−0.326
GI	GGTATC	1610.35	931	0.578	−0.548
GK	GGAAAA	3199.11	4553	1.423	0.353
GK	GGGAAG	4044.81	5674	1.403	0.338
GK	GGGAAA	3123.14	4119	1.319	0.277
GK	GGCAAG	5617.61	5712	1.017	0.017
GK	GGAAAG	4143.21	3706	0.894	−0.112
GK	GGCAAA	4337.55	3581	0.826	−0.192
GK	GGTAAA	2071.17	1334	0.644	−0.440
GK	GGTAAG	2682.40	540	0.201	−1.603
GL	GGCCTC	3017.19	4559	1.511	0.413
GL	GGTTTA	579.43	820	1.415	0.347
GL	GGTTTG	973.39	1294	1.329	0.285
GL	GGGCTG	4514.62	5878	1.302	0.264
GL	GGTCTT	993.42	1258	1.266	0.236
GL	GGCCTG	6270.10	7822	1.248	0.221
GL	GGGCTC	2172.45	2563	1.180	0.165
GL	GGATTA	894.98	991	1.107	0.102
GL	GGACTT	1534.44	1613	1.051	0.050
GL	GGCTTG	2038.53	2109	1.035	0.034
GL	GGCCTT	2080.48	2098	1.008	0.008
GL	GGACTA	827.51	799	0.966	−0.035
GL	GGGCTT	1497.99	1445	0.965	−0.036
GL	GGTCTC	1440.70	1365	0.947	−0.054
GL	GGTCTA	535.75	487	0.909	−0.095
GL	GGGCTA	807.86	726	0.899	−0.107
GL	GGCCTA	1121.99	968	0.863	−0.148
GL	GGCTTA	1213.47	935	0.771	−0.261
GL	GGACTC	2225.29	1656	0.744	−0.295
GL	GGATTG	1503.50	1062	0.706	−0.348
GL	GGTCTG	2993.96	2034	0.679	−0.387
GL	GGGTTG	1467.79	870	0.593	−0.523
GL	GGGTTA	873.73	467	0.534	−0.626
GL	GGACTG	4624.44	2384	0.516	−0.663
GM	GGCATG	3177.11	3953	1.244	0.219
GM	GGAATG	2343.24	2482	1.059	0.058
GM	GGGATG	2287.59	2247	0.982	−0.018
GM	GGTATG	1517.06	643	0.424	−0.858
GN	GGAAAT	2150.19	3332	1.550	0.438
GN	GGGAAC	2311.93	2816	1.218	0.197
GN	GGCAAC	3210.92	3701	1.153	0.142
GN	GGAAAC	2368.18	2679	1.131	0.123
GN	GGGAAT	2099.13	1823	0.868	−0.141
GN	GGCAAT	2915.36	2061	0.707	−0.347
GN	GGTAAT	1392.08	784	0.563	−0.574
GN	GGTAAC	1533.21	785	0.512	−0.669
GP	GGGCCC	2634.22	3947	1.498	0.404
GP	GGGCCG	954.47	1417	1.485	0.395
GP	GGCCCC	3658.52	4576	1.251	0.224
GP	GGCCCG	1325.61	1623	1.224	0.202
GP	GGTCCT	1564.62	1910	1.221	0.199
GP	GGGCCT	2359.31	2542	1.077	0.075
GP	GGTCCC	1746.93	1827	1.046	0.045
GP	GGCCCT	3276.71	2994	0.914	−0.090
GP	GGGCCA	2277.03	2003	0.880	−0.128
GP	GGTCCA	1510.06	1264	0.837	−0.178
GP	GGACCC	2698.30	2240	0.830	−0.186
GP	GGACCA	2332.42	1908	0.818	−0.201
GP	GGACCT	2416.70	1957	0.810	−0.211
GP	GGCCCA	3162.44	2548	0.806	−0.216
GP	GGTCCG	632.98	351	0.555	−0.590
GP	GGACCG	977.69	421	0.431	−0.843
GQ	GGACAA	1382.58	1677	1.213	0.193
GQ	GGGCAG	3769.06	4425	1.174	0.160
GQ	GGCCAG	5234.64	6081	1.162	0.150
GQ	GGTCAA	895.11	953	1.065	0.063
GQ	GGCCAA	1874.58	1593	0.850	−0.163
GQ	GGGCAA	1349.74	1124	0.833	−0.183
GQ	GGACAG	3860.75	3134	0.812	−0.209
GQ	GGTCAG	2499.53	1879	0.752	−0.285
GR	GGCCGC	1832.29	3615	1.973	0.680
GR	GGAAGA	1490.60	2294	1.539	0.431
GR	GGCCGG	2003.98	2892	1.443	0.367
GR	GGCCGT	784.47	1022	1.303	0.265
GR	GGTCGT	374.58	450	1.201	0.183
GR	GGCCGA	1086.32	1252	1.153	0.142
GR	GGGCGC	1319.29	1471	1.115	0.109
GR	GGTCGA	518.71	546	1.053	0.051
GR	GGCAGG	1984.13	2022	1.019	0.019
GR	GGGAGG	1428.62	1435	1.004	0.004
GR	GGGCGG	1442.91	1437	0.996	−0.004
GR	GGAAGG	1463.37	1370	0.936	−0.066
GR	GGGAGA	1455.20	1344	0.924	−0.079
GR	GGACGT	578.58	514	0.888	−0.118
GR	GGACGA	801.20	671	0.837	−0.177
GR	GGGCGT	564.84	471	0.834	−0.182
GR	GGCAGA	2021.05	1684	0.833	−0.182
GR	GGGCGA	782.17	626	0.800	−0.223
GR	GGTCGC	874.92	596	0.681	−0.384
GR	GGTCGG	956.90	555	0.580	−0.545
GR	GGTAGA	965.05	529	0.548	−0.601
GR	GGACGC	1351.39	729	0.539	−0.617
GR	GGACGG	1478.01	737	0.499	−0.696
GR	GGTAGG	947.42	244	0.258	−1.357
GS	GGCAGC	3581.32	6542	1.827	0.603
GS	GGCTCC	3161.05	5376	1.701	0.531
GS	GGCTCG	835.91	1323	1.583	0.459
GS	GGCAGT	2259.47	2875	1.272	0.241
GS	GGAAGT	1666.45	2085	1.251	0.224
GS	GGTTCT	1313.02	1563	1.190	0.174
GS	GGCTCT	2749.80	3087	1.123	0.116
GS	GGGAGC	2578.63	2566	0.995	−0.005
GS	GGTTCC	1509.39	1428	0.946	−0.055
GS	GGCTCA	2223.05	2101	0.945	−0.056
GS	GGTTCA	1061.50	981	0.924	−0.079
GS	GGAAGC	2641.36	2137	0.809	−0.212
GS	GGATCA	1639.59	1281	0.781	−0.247
GS	GGGAGT	1626.88	1267	0.779	−0.250
GS	GGATCT	2028.08	1470	0.725	−0.322
GS	GGGTCC	2276.03	1646	0.723	−0.324
GS	GGGTCT	1979.92	1280	0.646	−0.436
GS	GGGTCG	601.87	379	0.630	−0.463
GS	GGTAGT	1078.89	646	0.599	−0.513
GS	GGATCC	2331.40	1342	0.576	−0.552
GS	GGGTCA	1600.65	887	0.554	−0.590
GS	GGTTCG	399.14	209	0.524	−0.647
GS	GGATCG	616.51	276	0.448	−0.804
GS	GGTAGC	1710.07	723	0.423	−0.861
GT	GGCACC	3271.07	4870	1.489	0.398
GT	GGCACG	1074.53	1368	1.273	0.241
GT	GGGACC	2355.25	2817	1.196	0.179
GT	GGAACA	1953.05	2290	1.173	0.159
GT	GGAACT	1727.13	1900	1.100	0.095
GT	GGGACG	773.69	838	1.083	0.080
GT	GGGACA	1906.66	1903	0.998	−0.002
GT	GGCACT	2341.75	2331	0.995	−0.005
GT	GGCACA	2648.06	2499	0.944	−0.058
GT	GGGACT	1686.11	1534	0.910	−0.095
GT	GGAACC	2412.54	1841	0.763	−0.270
GT	GGTACT	1118.18	840	0.751	−0.286
GT	GGTACC	1561.93	994	0.636	−0.452
GT	GGTACA	1264.44	780	0.617	−0.483
GT	GGAACG	792.51	445	0.562	−0.577
GT	GGTACG	513.09	150	0.292	−1.230
GV	GGTGTT	816.93	1802	2.206	0.791
GV	GGTGTC	1045.94	2070	1.979	0.683
GV	GGTGTA	530.46	957	1.804	0.590
GV	GGTGTG	2069.18	3207	1.550	0.438
GV	GGAGTA	819.35	1225	1.495	0.402
GV	GGAGTT	1261.83	1841	1.459	0.378
GV	GGGGTC	1577.18	2150	1.363	0.310
GV	GGAGTC	1615.55	1839	1.138	0.130
GV	GGGGTT	1231.86	1123	0.912	−0.093
GV	GGGGTG	3120.14	2770	0.888	−0.119
GV	GGAGTG	3196.04	2641	0.826	−0.191
GV	GGGGTA	799.89	631	0.789	−0.237
GV	GGCGTC	2190.46	1653	0.755	−0.282
GV	GGCGTG	4333.39	2790	0.644	−0.440
GV	GGCGTT	1710.87	499	0.292	−1.232
GV	GGCGTA	1110.93	232	0.209	−1.566
GW	GGCTGG	2102.85	3748	1.782	0.578
GW	GGTTGG	1004.11	690	0.687	−0.375
GW	GGATGG	1550.94	1012	0.653	−0.427
GW	GGGTGG	1514.10	722	0.477	−0.741
GY	GGCTAC	2577.81	4581	1.777	0.575
GY	GGTTAT	1000.20	1309	1.309	0.269
GY	GGCTAT	2094.66	2528	1.207	0.188
GY	GGATAT	1544.90	1478	0.957	−0.044
GY	GGTTAC	1230.90	1074	0.873	−0.136
GY	GGATAC	1901.24	1052	0.553	−0.592
GY	GGGTAC	1856.09	982	0.529	−0.637
GY	GGGTAT	1508.21	710	0.471	−0.753
HA	CATGCT	1101.90	1959	1.778	0.575
HA	CATGCA	964.61	1670	1.731	0.549
HA	CATGCC	1675.52	2408	1.437	0.363
HA	CACGCG	624.72	681	1.090	0.086
HA	CATGCG	453.03	447	0.987	−0.013
HA	CACGCC	2310.52	1649	0.714	−0.337
HA	CACGCA	1330.18	617	0.464	−0.768
HA	CACGCT	1519.52	549	0.361	−1.018
HC	CACTGC	1778.65	2629	1.478	0.391
HC	CACTGT	1498.13	1717	1.146	0.136
HC	CATTGT	1086.40	673	0.619	−0.479
HC	CATTGC	1289.82	634	0.492	−0.710
HD	CATGAT	1329.76	2349	1.766	0.569
HD	CATGAC	1502.11	2329	1.550	0.439
HD	CACGAC	2071.40	1343	0.648	−0.433
HD	CACGAT	1833.73	716	0.390	−0.940
HE	CATGAA	1769.46	3512	1.985	0.686
HE	CATGAG	2366.33	3307	1.398	0.335
HE	CACGAG	3263.15	2230	0.683	−0.381
HE	CACGAA	2440.07	790	0.324	−1.128
HF	CACTTC	2538.66	3116	1.227	0.205
HF	CATTTT	1608.41	1806	1.123	0.116
HF	CACTTT	2217.98	1884	0.849	−0.163
HF	CATTTC	1840.95	1400	0.760	−0.274
HG	CATGGA	1246.72	2238	1.795	0.585
HG	CATGGT	807.15	1426	1.767	0.569
HG	CATGGG	1217.11	1849	1.519	0.418
HG	CATGGC	1690.37	2320	1.372	0.317
HG	CACGGC	2331.01	1680	0.721	−0.328
HG	CACGGG	1678.38	1184	0.705	−0.349
HG	CACGGT	1113.05	468	0.420	−0.866
HG	CACGGA	1719.21	638	0.371	−0.991
HH	CACCAC	2269.33	2795	1.232	0.208
HH	CATCAT	1193.37	1250	1.047	0.046
HH	CACCAT	1645.65	1453	0.883	−0.125
HH	CATCAC	1645.65	1256	0.763	−0.270
HI	CACATC	2433.52	3538	1.454	0.374
HI	CACATT	1924.40	1924	1.000	0.000
HI	CACATA	885.05	867	0.980	−0.021
HI	CATATT	1395.51	1260	0.903	−0.102
HI	CATATA	641.81	552	0.860	−0.151
HI	CATATC	1764.71	904	0.512	−0.669
HK	CACAAG	3102.81	3928	1.266	0.236
HK	CACAAA	2395.79	2432	1.015	0.015
HK	CATAAA	1737.35	1690	0.973	−0.028
HK	CATAAG	2250.06	1436	0.638	−0.449
HL	CATTTA	707.71	1053	1.488	0.397
HL	CATTTG	1188.90	1485	1.249	0.222
HL	CACCTG	5042.69	6030	1.196	0.179
HL	CACCTC	2426.56	2850	1.175	0.161
HL	CATCTT	1213.36	1409	1.161	0.149
HL	CACTTG	1639.48	1700	1.037	0.036
HL	CATCTA	654.36	649	0.992	−0.008
HL	CACCTT	1673.21	1499	0.896	−0.110
HL	CACCTA	902.35	761	0.843	−0.170
HL	CATCTC	1759.66	1422	0.808	−0.213
HL	CACTTA	975.93	781	0.800	−0.223
HL	CATCTG	3656.80	2202	0.602	−0.507
HM	CACATG	2348.18	3023	1.287	0.253
HM	CATATG	1702.82	1028	0.604	−0.505
HN	CACAAC	2031.88	2762	1.359	0.307
HN	CACAAT	1844.85	1832	0.993	−0.007
HN	CATAAT	1337.83	1225	0.916	−0.088
HN	CATAAC	1473.45	869	0.590	−0.528
HP	CACCCG	846.94	1341	1.583	0.460
HP	CATCCT	1518.15	1770	1.166	0.153
HP	CACCCC	2337.46	2530	1.082	0.079
HP	CATCCA	1465.21	1577	1.076	0.074
HP	CACCCA	2020.51	1919	0.950	−0.052
HP	CACCCT	2093.51	1859	0.888	−0.119
HP	CATCCC	1695.05	1265	0.746	−0.293
HP	CATCCG	614.18	330	0.537	−0.621
HQ	CATCAA	1143.96	1358	1.187	0.172
HQ	CACCAG	4405.09	4761	1.081	0.078
HQ	CATCAG	3194.43	2957	0.926	−0.077
HQ	CACCAA	1577.51	1245	0.789	−0.237
HR	CACAGG	1447.19	1936	1.338	0.291
HR	CACCGC	1336.44	1772	1.326	0.282
HR	CACAGA	1474.12	1788	1.213	0.193
HR	CACCGG	1461.67	1772	1.212	0.193
HR	CACCGT	572.18	667	1.166	0.153
HR	CATCGA	574.58	627	1.091	0.087
HR	CATCGT	414.93	452	1.089	0.086
HR	CACCGA	792.34	855	1.079	0.076
HR	CATCGG	1059.96	729	0.688	−0.374
HR	CATAGA	1068.98	635	0.594	−0.521
HR	CATCGC	969.15	565	0.583	−0.540
HR	CATAGG	1049.46	423	0.403	−0.909
HS	CACTCG	551.81	880	1.595	0.467
HS	CACAGC	2364.16	3726	1.576	0.455
HS	CACAGT	1491.56	1957	1.312	0.272
HS	CATTCA	1064.20	1307	1.228	0.206
HS	CATTCT	1316.36	1517	1.152	0.142
HS	CACTCC	2086.72	1964	0.941	−0.061
HS	CACTCA	1467.52	1318	0.898	−0.107
HS	CATTCC	1513.23	1219	0.806	−0.216
HS	CACTCT	1815.24	1231	0.678	−0.388
HS	CATAGT	1081.63	710	0.656	−0.421
HS	CATTCG	400.16	256	0.640	−0.447
HS	CATAGC	1714.41	782	0.456	−0.785
HT	CACACG	778.62	1526	1.960	0.673
HT	CACACT	1696.86	2036	1.200	0.182
HT	CACACA	1918.82	2255	1.175	0.161
HT	CACACC	2370.26	2537	1.070	0.068
HT	CATACT	1230.51	1306	1.061	0.060
HT	CATACA	1391.46	979	0.704	−0.352
HT	CATACC	1718.84	806	0.469	−0.757
HT	CATACG	564.63	225	0.398	−0.920
HV	CATGTT	869.32	1563	1.798	0.587
HV	CATGTA	564.48	880	1.559	0.444
HV	CATGTC	1113.00	1607	1.444	0.367
HV	CATGTG	2201.86	2797	1.270	0.239
HV	CACGTG	3036.34	2579	0.849	−0.163
HV	CACGTC	1534.82	1158	0.754	−0.282
HV	CACGTT	1198.78	434	0.362	−1.016
HV	CACGTA	778.41	279	0.358	−1.026
HW	CACTGG	1602.74	2197	1.371	0.315
HW	CATTGG	1162.26	568	0.489	−0.716
HY	CACTAC	1943.40	2385	1.227	0.205
HY	CATTAT	1145.15	1240	1.083	0.080
HY	CACTAT	1579.16	1378	0.873	−0.136
HY	CATTAC	1409.29	1074	0.762	−0.272
IA	ATTGCT	1886.56	3678	1.950	0.668
IA	ATAGCA	759.54	1446	1.904	0.644
IA	ATTGCA	1651.49	2818	1.706	0.534
IA	ATAGCT	867.65	1289	1.486	0.396
IA	ATTGCC	2868.63	3435	1.197	0.180
IA	ATAGCC	1319.32	1191	0.903	−0.102
IA	ATCGCG	980.82	708	0.722	−0.326
IA	ATCGCC	3627.56	2570	0.708	−0.345
IA	ATTGCG	775.62	494	0.637	−0.451
IA	ATAGCG	356.72	198	0.555	−0.589
IA	ATCGCA	2088.41	831	0.398	−0.922
IA	ATCGCT	2385.67	910	0.381	−0.964
IC	ATCTGC	2115.05	3055	1.444	0.368
IC	ATCTGT	1781.48	2074	1.164	0.152
IC	ATATGT	647.91	731	1.128	0.121
IC	ATTTGT	1408.77	1197	0.850	−0.163
IC	ATATGC	769.23	470	0.611	−0.493
IC	ATTTGC	1672.56	868	0.519	−0.656
ID	ATTGAT	2604.76	4341	1.667	0.511
ID	ATAGAT	1197.96	1947	1.625	0.486
ID	ATTGAC	2942.37	3938	1.338	0.291
ID	ATAGAC	1353.23	1476	1.091	0.087
ID	ATCGAC	3720.81	2270	0.610	−0.494
ID	ATCGAT	3293.87	1141	0.346	−1.060
IE	ATAGAA	1371.51	2939	2.143	0.762
IE	ATTGAA	2982.12	5518	1.850	0.615
IE	ATTGAG	3988.04	4634	1.162	0.150
IE	ATAGAG	1834.15	1898	1.035	0.034
IE	ATCGAG	5043.12	3007	0.596	−0.517
IE	ATCGAA	3771.07	994	0.264	−1.333
IF	ATATTT	1144.73	1929	1.685	0.522
IF	ATCTTC	3602.60	4836	1.342	0.294
IF	ATTTTT	2489.02	2226	0.894	−0.112
IF	ATCTTT	3147.52	2779	0.883	−0.125
IF	ATATTC	1310.24	886	0.676	−0.391
IF	ATTTTC	2848.89	1887	0.662	−0.412
IG	ATTGGT	1013.16	2102	2.075	0.730
IG	ATTGGA	1564.91	3151	2.014	0.700
IG	ATAGGA	719.72	1054	1.464	0.381
IG	ATTGGG	1527.75	2144	1.403	0.339
IG	ATAGGT	465.96	596	1.279	0.246
IG	ATTGGC	2121.81	2706	1.275	0.243
IG	ATAGGG	702.63	549	0.781	−0.247
IG	ATAGGC	975.84	700	0.717	−0.332
IG	ATCGGG	1931.93	1244	0.644	−0.440
IG	ATCGGC	2683.15	1619	0.603	−0.505
IG	ATCGGT	1281.20	498	0.389	−0.945
IG	ATCGGA	1978.93	604	0.305	−1.187
IH	ATTCAT	1622.93	2242	1.381	0.323
IH	ATCCAC	2830.09	3367	1.190	0.174
IH	ATACAT	746.40	760	1.018	0.018
IH	ATCCAT	2052.29	1814	0.884	−0.123
IH	ATTCAC	2238.00	1778	0.794	−0.230
IH	ATACAC	1029.28	558	0.542	−0.612
II	ATCATC	3797.03	5979	1.575	0.454
II	ATAATA	502.24	700	1.394	0.332
II	ATAATT	1092.04	1309	1.199	0.181
II	ATCATT	3002.64	3321	1.106	0.101
II	ATTATT	2374.46	2157	0.908	−0.096
II	ATCATA	1380.95	1183	0.857	−0.155
II	ATTATA	1092.04	921	0.843	−0.170
II	ATAATC	1380.95	715	0.518	−0.658
II	ATTATC	3002.64	1340	0.446	−0.807
IK	ATAAAA	1419.09	2244	1.581	0.458
IK	ATCAAG	5053.39	5884	1.164	0.152
IK	ATAAAG	1837.88	1943	1.057	0.056
IK	ATTAAA	3085.58	3107	1.007	0.007
IK	ATCAAA	3901.90	3830	0.982	−0.019
IK	ATTAAG	3996.16	2286	0.572	−0.559
IL	ATTTTA	977.08	1679	1.718	0.541
IL	ATATTA	449.37	723	1.609	0.476
IL	ATTTTG	1641.41	2339	1.425	0.354
IL	ATTCTT	1675.18	2271	1.356	0.304
IL	ATCCTC	3072.14	4017	1.308	0.268
IL	ATCCTG	6384.29	7754	1.215	0.194
IL	ATTCTA	903.41	1021	1.130	0.122
IL	ATCTTG	2075.66	2250	1.084	0.081
IL	ATCCTA	1142.42	1170	1.024	0.024
IL	ATACTA	415.49	416	1.001	0.001
IL	ATCCTT	2118.37	2058	0.972	−0.029
IL	ATATTG	754.90	717	0.950	−0.052
IL	ATACTT	770.44	726	0.942	−0.059
IL	ATCTTA	1235.57	1077	0.872	−0.137
IL	ATTCTC	2429.41	1918	0.789	−0.236
IL	ATTCTG	5048.62	3005	0.595	−0.519
IL	ATACTC	1117.32	458	0.410	−0.892
IL	ATACTG	2321.92	934	0.402	−0.911
IM	ATCATG	3206.80	4314	1.345	0.297
IM	ATAATG	1166.29	1196	1.025	0.025
IM	ATTATG	2535.90	1399	0.552	−0.595
IN	ATAAAT	1088.42	1649	1.515	0.415
IN	ATCAAC	3296.07	4599	1.395	0.333
IN	ATCAAT	2992.68	2890	0.966	−0.035
IN	ATAAAC	1198.76	1113	0.928	−0.074
IN	ATTAAT	2366.58	1967	0.831	−0.185
IN	ATTAAC	2606.49	1331	0.511	−0.672
IP	ATTCCT	2051.78	2787	1.358	0.306
IP	ATTCCA	1980.23	2644	1.335	0.289
IP	ATACCA	910.73	1047	1.150	0.139
IP	ATCCCC	2896.94	3229	1.115	0.109
IP	ATACCT	943.64	995	1.054	0.053
IP	ATCCCG	1049.66	1073	1.022	0.022
IP	ATCCCA	2504.13	2366	0.945	−0.057
IP	ATCCCT	2594.61	2451	0.945	−0.057
IP	ATTCCC	2290.86	1775	0.775	−0.255
IP	ATACCC	1053.60	610	0.579	−0.547
IP	ATTCCG	830.06	386	0.465	−0.766
IP	ATACCG	381.76	125	0.327	−1.116
IQ	ATACAA	765.47	950	1.241	0.216
IQ	ATTCAA	1664.38	2045	1.229	0.206
IQ	ATCCAG	5877.26	6881	1.171	0.158
IQ	ATTCAG	4647.67	3987	0.858	−0.153
IQ	ATCCAA	2104.71	1765	0.839	−0.176
IQ	ATACAG	2137.52	1569	0.734	−0.309
IR	ATCCGC	1552.18	2623	1.690	0.525
IR	ATTCGA	727.72	1142	1.569	0.451
IR	ATCCGA	920.25	1434	1.558	0.444
IR	ATCCGT	664.55	943	1.419	0.350
IR	ATAAGA	622.67	877	1.408	0.342
IR	ATCCGG	1697.63	2265	1.334	0.288
IR	ATTCGT	525.51	677	1.288	0.253
IR	ATCAGA	1712.09	1680	0.981	−0.019
IR	ATCAGG	1680.81	1513	0.900	−0.105
IR	ATAAGG	611.30	547	0.895	−0.111
IR	ATACGT	241.69	213	0.881	−0.126
IR	ATACGA	334.69	292	0.872	−0.136
IR	ATTCGG	1342.46	907	0.676	−0.392
IR	ATTAGA	1353.90	900	0.665	−0.408
IR	ATTCGC	1227.45	780	0.635	−0.453
IR	ATACGG	617.42	260	0.421	−0.865
IR	ATTAGG	1329.16	503	0.378	−0.972
IR	ATACGC	564.52	170	0.301	−1.200
IS	ATCTCC	2689.59	3743	1.392	0.330
IS	ATATCA	687.92	954	1.387	0.327
IS	ATCAGC	3047.17	3998	1.312	0.272
IS	ATTTCT	1850.19	2423	1.310	0.270
IS	ATTTCA	1495.77	1957	1.308	0.269
IS	ATCAGT	1922.48	2287	1.190	0.174
IS	ATATCT	850.92	1012	1.189	0.173
IS	ATCTCG	711.23	773	1.087	0.083
IS	ATAAGT	699.19	695	0.994	−0.006
IS	ATCTCT	2339.68	2317	0.990	−0.010
IS	ATCTCA	1891.49	1767	0.934	−0.068
IS	ATTTCC	2126.89	1795	0.844	−0.170
IS	ATATCC	978.18	703	0.719	−0.330
IS	ATTAGT	1520.28	906	0.596	−0.518
IS	ATAAGC	1108.24	636	0.574	−0.555
IS	ATATCG	258.67	132	0.510	−0.673
IS	ATTTCG	562.43	255	0.453	−0.791
IS	ATTAGC	2409.67	797	0.331	−1.106
IT	ATCACC	3094.94	4722	1.526	0.422
IT	ATCACG	1016.68	1306	1.285	0.250
IT	ATAACT	805.82	1009	1.252	0.225
IT	ATCACT	2215.66	2751	1.242	0.216
IT	ATCACA	2505.48	2989	1.193	0.176
IT	ATAACA	911.22	1079	1.184	0.169
IT	ATTACT	1752.12	1369	0.781	−0.247
IT	ATTACA	1981.30	1531	0.773	−0.258
IT	ATAACC	1125.61	741	0.658	−0.418
IT	ATAACG	369.76	204	0.552	−0.595
IT	ATTACC	2447.44	1083	0.443	−0.815
IT	ATTACG	803.98	246	0.306	−1.184
IV	ATTGTT	1261.28	2414	1.914	0.649
IV	ATTGTA	819.00	1478	1.805	0.590
IV	ATAGTA	376.67	645	1.712	0.538
IV	ATAGTT	580.08	877	1.512	0.413
IV	ATTGTC	1614.84	2315	1.434	0.360
IV	ATTGTG	3194.65	3762	1.178	0.163
IV	ATCGTC	2042.07	1679	0.822	−0.196
IV	ATAGTG	1469.26	1196	0.814	−0.206
IV	ATAGTC	742.69	575	0.774	−0.256
IV	ATCGTG	4039.83	2922	0.723	−0.324
IV	ATCGTA	1035.67	361	0.349	−1.054
IV	ATCGTT	1594.97	547	0.343	−1.070
IW	ATCTGG	1887.23	2427	1.286	0.252
IW	ATATGG	686.37	622	0.906	−0.098
IW	ATTTGG	1492.40	1017	0.681	−0.384
IY	ATCTAC	2708.47	3486	1.287	0.252
IY	ATATAT	800.43	953	1.191	0.174
IY	ATTTAT	1740.39	1984	1.140	0.131
IY	ATCTAT	2200.83	2196	0.998	−0.002
IY	ATTTAC	2141.83	1403	0.655	−0.423
IY	ATATAC	985.05	555	0.563	−0.574
KA	AAAGCA	3029.93	4322	1.426	0.355
KA	AAAGCT	3461.21	4262	1.231	0.208
KA	AAGGCC	6816.15	6676	0.979	−0.021
KA	AAGGCG	1842.96	1790	0.971	−0.029
KA	AAGGCA	3924.10	3654	0.931	−0.071
KA	AAAGCC	5262.99	4742	0.901	−0.104
KA	AAGGCT	4482.65	4032	0.899	−0.106
KA	AAAGCG	1423.01	765	0.538	−0.621
KC	AAATGT	1815.55	2671	1.471	0.386
KC	AAGTGT	2351.33	2267	0.964	−0.037
KC	AAGTGC	2791.62	2498	0.895	−0.111
KC	AAATGC	2155.50	1678	0.778	−0.250
KD	AAAGAT	4684.00	6115	1.306	0.267
KD	AAGGAC	6852.58	6836	0.998	−0.002
KD	AAGGAT	6066.30	5379	0.887	−0.120
KD	AAAGAC	5291.12	4564	0.863	−0.148
KE	AAAGAA	6989.41	9895	1.416	0.348
KE	AAGGAG	12105.47	12287	1.015	0.015
KE	AAGGAA	9052.06	8366	0.924	−0.079
KE	AAAGAG	9347.06	6946	0.743	−0.297
KF	AAATTT	2631.62	3140	1.193	0.177
KF	AAGTTT	3408.25	3638	1.067	0.065
KF	AAGTTC	3901.02	3950	1.013	0.012
KF	AAATTC	3012.11	2225	0.739	−0.303
KG	AAAGGA	2672.15	4509	1.687	0.523
KG	AAAGGT	1730.00	2402	1.388	0.328
KG	AAAGGC	3623.06	3435	0.948	−0.053
KG	AAAGGG	2608.69	2465	0.945	−0.057
KG	AAGGGC	4692.27	4309	0.918	−0.085
KG	AAGGGT	2240.55	1978	0.883	−0.125
KG	AAGGGG	3378.54	2740	0.811	−0.209
KG	AAGGGA	3460.73	2568	0.742	−0.298
KH	AAACAT	1929.29	2356	1.221	0.200
KH	AAGCAC	3445.60	3583	1.040	0.039
KH	AAGCAT	2498.64	2430	0.973	−0.028
KH	AAACAC	2660.47	2165	0.814	−0.206
KI	AAAATA	1547.96	2667	1.723	0.544
KI	AAAATT	3365.76	3894	1.157	0.146
KI	AAGATC	5512.26	5523	1.002	0.002
KI	AAGATA	2004.77	1943	0.969	−0.031
KI	AAGATT	4359.03	3732	0.856	−0.155
KI	AAAATC	4256.21	3287	0.772	−0.258
KK	AAGAAG	11070.03	13815	1.248	0.222
KK	AAGAAA	8547.55	10129	1.185	0.170
KK	AAAAAG	8547.55	6145	0.719	−0.330
KK	AAAAAA	6599.86	4676	0.708	−0.345
KL	AAATTA	1273.72	2084	1.636	0.492
KL	AAACTA	1177.70	1750	1.486	0.396
KL	AAACTT	2183.78	3014	1.380	0.322
KL	AAGCTG	8523.68	9600	1.126	0.119
KL	AAGCTA	1525.25	1660	1.088	0.085
KL	AAGCTC	4101.62	4076	0.994	−0.006
KL	AAATTG	2139.75	2113	0.987	−0.013
KL	AAGCTT	2828.24	2772	0.980	−0.020
KL	AAGTTA	1649.61	1459	0.884	−0.123
KL	AAACTC	3167.00	2653	0.838	−0.177
KL	AAGTTG	2771.21	2280	0.823	−0.195
KL	AAACTG	6581.43	4462	0.678	−0.389
KM	AAGATG	5479.27	5650	1.031	0.031
KM	AAAATG	4230.73	4060	0.960	−0.041
KN	AAAAAT	3683.47	4378	1.189	0.173
KN	AAGAAC	5254.13	5515	1.050	0.048
KN	AAGAAT	4770.51	4618	0.968	−0.032
KN	AAAAAC	4056.89	3254	0.802	−0.221
KP	AAACCA	2803.51	3370	1.202	0.184
KP	AAGCCC	4200.41	4673	1.113	0.107
KP	AAGCCA	3630.85	4035	1.111	0.106
KP	AAACCT	2904.80	3118	1.073	0.071
KP	AAGCCG	1521.96	1544	1.014	0.014
KP	AAGCCT	3762.04	3396	0.903	−0.102
KP	AAACCC	3243.28	2624	0.809	−0.212
KP	AAACCG	1175.16	482	0.410	−0.891
KQ	AAACAA	2178.87	3274	1.503	0.407
KQ	AAGCAA	2821.88	3177	1.126	0.119
KQ	AAGCAG	7879.90	8081	1.026	0.025
KQ	AAACAG	6084.35	4433	0.729	−0.317
KR	AAAAGA	2247.57	3147	1.400	0.337
KR	AAGAGG	2857.67	3975	1.391	0.330
KR	AAGAGA	2910.85	3511	1.206	0.187
KR	AAAAGG	2206.51	2325	1.054	0.052
KR	AAACGT	872.39	862	0.988	−0.012
KR	AAGCGG	2886.27	2828	0.980	−0.020
KR	AAGCGC	2638.99	2532	0.959	−0.041
KR	AAACGA	1208.07	1087	0.900	−0.106
KR	AAGCGT	1129.84	978	0.866	−0.144
KR	AAGCGA	1564.59	1325	0.847	−0.166
KR	AAACGG	2228.59	1178	0.529	−0.638
KR	AAACGC	2037.65	1041	0.511	−0.672
KS	AAATCA	1871.14	2533	1.354	0.303
KS	AAAAGT	1901.80	2389	1.256	0.228
KS	AAATCT	2314.50	2793	1.207	0.188
KS	AAGTCA	2423.33	2566	1.059	0.057
KS	AAGAGC	3903.97	4045	1.036	0.035
KS	AAGAGT	2463.04	2459	0.998	−0.002
KS	AAGTCG	911.22	904	0.992	−0.008
KS	AAGTCC	3445.84	3100	0.900	−0.106
KS	AAGTCT	2997.54	2675	0.892	−0.114
KS	AAATCC	2660.65	2304	0.866	−0.144
KS	AAAAGC	3014.39	2381	0.790	−0.236
KS	AAATCG	703.58	462	0.657	−0.421
KT	AAAACA	2831.74	3611	1.275	0.243
KT	AAGACG	1488.17	1790	1.203	0.185
KT	AAAACT	2504.18	2969	1.186	0.170
KT	AAGACC	4530.26	4475	0.988	−0.012
KT	AAGACA	3667.42	3574	0.975	−0.026
KT	AAGACT	3243.20	2876	0.887	−0.120
KT	AAAACC	3497.97	2854	0.816	−0.203
KT	AAAACG	1149.07	763	0.664	−0.409
KV	AAAGTA	1317.00	2214	1.681	0.519
KV	AAAGTT	2028.22	3042	1.500	0.405
KV	AAAGTC	2596.78	2642	1.017	0.017
KV	AAGGTG	6653.25	6512	0.979	−0.021
KV	AAGGTC	3363.11	3016	0.897	−0.109
KV	AAGGTT	2626.77	2294	0.873	−0.135
KV	AAAGTG	5137.21	4417	0.860	−0.151
KV	AAGGTA	1705.66	1291	0.757	−0.279
KW	AAGTGG	2598.56	2701	1.039	0.039
KW	AAATGG	2006.44	1904	0.949	−0.052
KY	AAATAT	2319.32	2982	1.286	0.251
KY	AAGTAC	3696.62	3603	0.975	−0.026
KY	AAATAC	2854.29	2763	0.968	−0.033
KY	AAGTAT	3003.78	2526	0.841	−0.173
LA	CTGGCG	2275.39	3643	1.601	0.471
LA	TTGGCA	1575.16	2350	1.492	0.400
LA	CTGGCC	8415.49	12456	1.480	0.392
LA	TTGGCT	1799.36	2643	1.469	0.384
LA	TTAGCA	937.64	1314	1.401	0.337
LA	CTTGCT	1836.39	2345	1.277	0.244
LA	CTAGCA	866.95	1107	1.277	0.244
LA	CTTGCA	1607.57	1861	1.158	0.146
LA	TTAGCT	1071.10	1239	1.157	0.146
LA	CTGGCT	5534.46	6333	1.144	0.135
LA	CTAGCT	990.35	1099	1.110	0.104
LA	CTGGCA	4844.85	5013	1.035	0.034
LA	TTGGCC	2736.04	2824	1.032	0.032
LA	TTGGCG	739.77	623	0.842	−0.172
LA	CTTGCC	2792.34	2201	0.788	−0.238
LA	CTAGCC	1505.89	1159	0.770	−0.262
LA	CTAGCG	407.16	253	0.621	−0.476
LA	TTAGCC	1628.68	941	0.578	−0.549
LA	CTTGCG	755.00	346	0.458	−0.780
LA	TTAGCG	440.36	198	0.450	−0.799
LA	CTCGCC	4049.56	1527	0.377	−0.975
LA	CTCGCG	1094.93	390	0.356	−1.032
LA	CTCGCT	2663.20	605	0.227	−1.482
LA	CTCGCA	2331.36	429	0.184	−1.693
LC	CTCTGC	1769.27	3523	1.991	0.689
LC	CTCTGT	1490.23	2145	1.439	0.364
LC	CTTTGT	1027.58	1155	1.124	0.117
LC	TTATGT	599.35	627	1.046	0.045
LC	CTGTGC	3676.77	3517	0.957	−0.044
LC	TTGTGT	1006.86	856	0.850	−0.162
LC	CTTTGC	1219.99	974	0.798	−0.225
LC	CTGTGT	3096.89	2370	0.765	−0.268
LC	CTATGT	554.17	417	0.752	−0.284
LC	TTGTGC	1195.39	722	0.604	−0.504
LC	TTATGC	711.58	368	0.517	−0.659
LC	CTATGC	657.93	332	0.505	−0.684
LD	TTGGAT	2174.51	3688	1.696	0.528
LD	TTAGAT	1294.41	1977	1.527	0.424
LD	CTGGAC	7555.23	10531	1.394	0.332
LD	CTAGAT	1196.83	1584	1.323	0.280
LD	TTGGAC	2456.35	2775	1.130	0.122
LD	CTTGAT	2219.25	2463	1.110	0.104
LD	CTGGAT	6688.33	6912	1.033	0.033
LD	CTAGAC	1351.95	1390	1.028	0.028
LD	CTTGAC	2506.90	1832	0.731	−0.314
LD	TTAGAC	1462.19	969	0.663	−0.411
LD	CTCGAC	3635.60	981	0.270	−1.310
LD	CTCGAT	3218.44	658	0.204	−1.587
LE	TTAGAA	1739.66	3085	1.773	0.573
LE	CTAGAA	1608.51	2701	1.679	0.518
LE	TTGGAA	2922.49	4652	1.592	0.465
LE	CTGGAG	12021.09	18044	1.501	0.406
LE	TTGGAG	3908.29	4774	1.222	0.200
LE	CTAGAG	2151.09	2515	1.169	0.156
LE	CTTGAA	2982.63	3161	1.060	0.058
LE	CTGGAA	8988.96	7642	0.850	−0.162
LE	TTAGAG	2326.48	1873	0.805	−0.217
LE	CTTGAG	3988.72	2484	0.623	−0.474
LE	CTCGAG	5784.58	1305	0.226	−1.489
LE	CTCGAA	4325.51	512	0.118	−2.134
LF	CTCTTC	2629.18	6495	2.470	0.904
LF	TTATTT	923.85	1405	1.521	0.419
LF	CTCTTT	2297.07	3446	1.500	0.406
LF	CTTTTT	1583.93	1937	1.223	0.201
LF	CTTTTC	1812.93	1936	1.068	0.066
LF	CTATTT	854.20	876	1.026	0.025
LF	TTGTTT	1551.99	1544	0.995	−0.005
LF	CTGTTT	4773.59	2957	0.619	−0.479
LF	CTGTTC	5463.77	3119	0.571	−0.561
LF	TTATTC	1057.42	583	0.551	−0.595
LF	TTGTTC	1776.38	940	0.529	−0.636
LF	CTATTC	977.70	464	0.475	−0.745
LG	CTTGGA	1534.14	2667	1.738	0.553
LG	CTTGGT	993.23	1579	1.590	0.464
LG	CTGGGC	6268.87	9794	1.562	0.446
LG	CTAGGA	827.35	1087	1.314	0.273
LG	CTTGGG	1497.70	1881	1.256	0.228
LG	TTAGGA	894.81	1114	1.245	0.219
LG	CTGGGG	4513.74	5602	1.241	0.216
LG	TTGGGT	973.20	1194	1.227	0.204
LG	TTGGGA	1503.20	1820	1.211	0.191
LG	CTAGGT	535.64	611	1.141	0.132
LG	TTAGGT	579.32	611	1.055	0.053
LG	TTGGGG	1467.50	1452	0.989	−0.011
LG	CTGGGT	2993.37	2947	0.985	−0.016
LG	CTTGGC	2080.08	2009	0.966	−0.035
LG	CTAGGG	807.70	766	0.948	−0.053
LG	TTGGGC	2038.13	1786	0.876	−0.132
LG	CTGGGA	4623.54	4034	0.872	−0.136
LG	CTAGGC	1121.77	940	0.838	−0.177
LG	TTAGGG	873.56	529	0.606	−0.502
LG	CTCGGG	2172.02	1076	0.495	−0.702
LG	CTCGGC	3016.60	1313	0.435	−0.832
LG	TTAGGC	1213.24	507	0.418	−0.873
LG	CTCGGT	1440.42	365	0.253	−1.373
LG	CTCGGA	2224.86	510	0.229	−1.473
LH	CTTCAT	1127.31	1980	1.756	0.563
LH	TTACAT	657.52	935	1.422	0.352
LH	CTACAT	607.95	741	1.219	0.198
LH	CTGCAC	4685.05	5459	1.165	0.153
LH	CTCCAC	2254.46	2204	0.978	−0.023
LH	CTTCAC	1554.55	1490	0.958	−0.042
LH	CTCCAT	1634.86	1521	0.930	−0.072
LH	CTACAC	838.36	777	0.927	−0.076
LH	TTGCAT	1104.58	1017	0.921	−0.083
LH	TTGCAC	1523.20	1140	0.748	−0.290
LH	CTGCAT	3397.45	2394	0.705	−0.350
LH	TTACAC	906.71	634	0.699	−0.358
LI	CTCATC	2602.42	6250	2.402	0.876
LI	TTAATA	380.66	798	2.096	0.740
LI	TTAATT	827.68	1290	1.559	0.444
LI	CTCATT	2057.96	3117	1.515	0.415
LI	CTAATA	351.96	516	1.466	0.383
LI	CTAATT	765.28	952	1.244	0.218
LI	CTTATT	1419.05	1761	1.241	0.216
LI	TTGATA	639.48	791	1.237	0.213
LI	TTGATT	1390.44	1468	1.056	0.054
LI	CTTATA	652.64	683	1.047	0.045
LI	CTCATA	946.48	919	0.971	−0.029
LI	CTTATC	1794.48	1189	0.663	−0.412
LI	TTGATC	1758.29	1135	0.646	−0.438
LI	CTGATC	5408.15	3356	0.621	−0.477
LI	CTGATT	4276.70	2639	0.617	−0.483
LI	CTGATA	1966.91	1193	0.607	−0.500
LI	TTAATC	1046.66	633	0.605	−0.503
LI	CTAATC	967.75	563	0.582	−0.542
LK	TTAAAA	1429.91	2557	1.788	0.581
LK	CTAAAA	1322.10	1842	1.393	0.332
LK	TTGAAA	2402.12	3193	1.329	0.285
LK	CTCAAG	4604.55	6048	1.313	0.273
LK	CTAAAG	1712.27	2078	1.214	0.194
LK	TTAAAG	1851.89	2128	1.149	0.139
LK	CTGAAG	9568.82	10212	1.067	0.065
LK	TTGAAG	3111.01	3222	1.036	0.035
LK	CTCAAA	3555.33	2768	0.779	−0.250
LK	CTTAAA	2451.55	1850	0.755	−0.282
LK	CTGAAA	7388.42	5227	0.707	−0.346
LK	CTTAAG	3175.03	1448	0.456	−0.785
LL	TTATTA	500.55	802	1.602	0.471
LL	CTTCTA	793.49	1132	1.427	0.355
LL	CTTCTT	1471.36	2099	1.427	0.355
LL	CTTTTA	858.19	1203	1.402	0.338
LL	CTGCTG	13364.10	18236	1.365	0.311
LL	CTTTTG	1441.69	1945	1.349	0.299
LL	TTACTA	462.82	608	1.314	0.273
LL	CTCCTC	3094.54	3800	1.228	0.205
LL	CTCCTG	6430.85	7786	1.211	0.191
LL	TTACTT	858.19	1039	1.211	0.191
LL	TTGCTA	777.49	929	1.195	0.178
LL	CTGCTC	6430.85	7550	1.174	0.160
LL	CTACTA	427.93	474	1.108	0.102
LL	CTTCTC	2133.82	2292	1.074	0.072
LL	CTACTT	793.49	839	1.057	0.056
LL	CTCTTG	2090.79	2131	1.019	0.019
LL	TTGCTT	1441.69	1464	1.015	0.015
LL	TTATTG	840.89	818	0.973	−0.028
LL	CTCCTT	2133.82	2034	0.953	−0.048
LL	TTGTTA	840.89	771	0.917	−0.087
LL	TTGTTG	1412.62	1289	0.912	−0.092
LL	CTCCTA	1150.75	1034	0.899	−0.107
LL	TTGCTG	4344.93	3820	0.879	−0.129
LL	CTTCTG	4434.34	3837	0.865	−0.145
LL	CTGCTA	2391.41	1913	0.800	−0.223
LL	CTCTTA	1244.58	959	0.771	−0.261
LL	CTATTA	462.82	354	0.765	−0.268
LL	CTGCTT	4434.34	3148	0.710	−0.343
LL	TTGCTC	2090.79	1440	0.689	−0.373
LL	CTACTC	1150.75	792	0.688	−0.374
LL	CTATTG	777.49	532	0.684	−0.379
LL	CTACTG	2391.41	1583	0.662	−0.413
LL	CTGTTG	4344.93	2615	0.602	−0.508
LL	TTACTC	1244.58	657	0.528	−0.639
LL	TTACTG	2586.40	1358	0.525	−0.644
LL	CTGTTA	2586.40	953	0.368	−0.998
LM	CTCATG	2631.41	4030	1.531	0.426
LM	TTAATG	1058.32	1228	1.160	0.149
LM	CTAATG	978.53	1101	1.125	0.118
LM	TTGATG	1777.88	1763	0.992	−0.008
LM	CTGATG	5468.39	4470	0.817	−0.202
LM	CTTATG	1814.47	1137	0.627	−0.467
LN	TTAAAT	962.36	1926	2.001	0.694
LN	CTCAAC	2635.40	4681	1.776	0.574
LN	CTAAAT	889.81	1446	1.625	0.486
LN	TTGAAT	1616.68	2048	1.267	0.236
LN	CTCAAT	2392.82	2652	1.108	0.103
LN	CTAAAC	980.01	922	0.941	−0.061
LN	TTAAAC	1059.92	965	0.910	−0.094
LN	CTTAAT	1649.95	1441	0.873	−0.135
LN	TTGAAC	1780.58	1541	0.865	−0.145
LN	CTGAAC	5476.68	4308	0.787	−0.240
LN	CTGAAT	4972.58	3413	0.686	−0.376
LN	CTTAAC	1817.22	891	0.490	−0.713
LP	CTTCCT	1728.14	2795	1.617	0.481
LP	CTTCCA	1667.88	2369	1.420	0.351
LP	CTGCCC	5815.10	7856	1.351	0.301
LP	TTACCT	1007.96	1244	1.234	0.210
LP	CTGCCG	2107.02	2489	1.181	0.167
LP	TTACCA	972.81	1140	1.172	0.159
LP	CTCCCG	1013.90	1184	1.168	0.155
LP	TTGCCA	1634.25	1897	1.161	0.149
LP	CTACCT	931.97	1045	1.121	0.114
LP	TTGCCT	1693.30	1800	1.063	0.061
LP	CTTCCC	1929.51	1889	0.979	−0.021
LP	CTACCA	899.47	850	0.945	−0.057
LP	CTCCCA	2418.82	2126	0.879	−0.129
LP	CTGCCT	5208.23	4563	0.876	−0.132
LP	CTCCCT	2506.21	2192	0.875	−0.134
LP	CTACCC	1040.57	888	0.853	−0.159
LP	CTCCCC	2798.25	2369	0.847	−0.167
LP	TTGCCC	1890.60	1560	0.825	−0.192
LP	TTGCCG	685.03	478	0.698	−0.360
LP	CTGCCA	5026.60	3348	0.666	−0.406
LP	CTTCCG	699.13	451	0.645	−0.438
LP	TTACCC	1125.42	666	0.592	−0.525
LP	CTACCG	377.04	211	0.560	−0.580
LP	TTACCG	407.78	175	0.429	−0.846
LQ	TTACAA	864.28	1290	1.493	0.401
LQ	CTACAA	799.12	1188	1.487	0.397
LQ	CTTCAA	1481.79	2098	1.416	0.348
LQ	CTACAG	2231.48	2674	1.198	0.181
LQ	CTGCAG	12470.36	14508	1.163	0.151
LQ	CTTCAG	4137.79	4363	1.054	0.053
LQ	TTGCAA	1451.91	1467	1.010	0.010
LQ	CTCCAG	6000.78	5430	0.905	−0.100
LQ	TTACAG	2413.43	2107	0.873	−0.136
LQ	TTGCAG	4054.36	3177	0.784	−0.244
LQ	CTCCAA	2148.94	1524	0.709	−0.344
LQ	CTGCAA	4465.77	2694	0.603	−0.505
LR	CTTCGA	661.43	1365	2.064	0.725
LR	CTTCGT	477.64	784	1.641	0.496
LR	CTGCGG	3677.31	5467	1.487	0.397
LR	TTAAGA	717.74	1026	1.429	0.357
LR	CTGCGC	3362.26	4574	1.360	0.308
LR	CTCCGA	959.23	1289	1.344	0.295
LR	CTCCGG	1769.53	2229	1.260	0.231
LR	CTAAGA	663.63	821	1.237	0.213
LR	CTCAGG	1752.00	2047	1.168	0.156
LR	CTTCGG	1220.17	1415	1.160	0.148
LR	CTCCGT	692.69	771	1.113	0.107
LR	TTACGA	385.79	427	1.107	0.101
LR	CTAAGG	651.51	721	1.107	0.101
LR	CTCCGC	1617.93	1790	1.106	0.101
LR	TTGAGA	1205.75	1290	1.070	0.068
LR	CTACGT	257.59	275	1.068	0.065
LR	CTACGA	356.70	378	1.060	0.058
LR	CTGAGG	3640.88	3637	0.999	−0.001
LR	TTAAGG	704.63	678	0.962	−0.039
LR	TTACGT	278.59	264	0.948	−0.054
LR	CTGCGT	1439.50	1363	0.947	−0.055
LR	TTGAGG	1183.72	1080	0.912	−0.092
LR	CTACGG	658.03	577	0.877	−0.131
LR	CTCAGA	1784.60	1469	0.823	−0.195
LR	CTTCGC	1115.63	819	0.734	−0.309
LR	CTACGC	601.65	438	0.728	−0.317
LR	CTGCGA	1993.40	1399	0.702	−0.354
LR	TTGCGT	468.01	321	0.686	−0.377
LR	CTGAGA	3708.63	2486	0.670	−0.400
LR	TTGCGG	1195.56	772	0.646	−0.437
LR	TTGCGA	648.09	418	0.645	−0.439
LR	CTTAGA	1230.56	694	0.564	−0.573
LR	TTACGG	711.68	383	0.538	−0.620
LR	TTGCGC	1093.14	542	0.496	−0.702
LR	CTTAGG	1208.08	503	0.416	−0.876
LR	TTACGC	650.71	232	0.357	−1.031
LS	CTCAGC	2740.30	5167	1.886	0.634
LS	CTTTCT	1450.83	2502	1.725	0.545
LS	CTCTCC	2418.72	4070	1.683	0.520
LS	CTCTCG	639.61	1016	1.588	0.463
LS	CTCAGT	1728.87	2589	1.498	0.404
LS	TTATCA	684.12	963	1.408	0.342
LS	TTATCT	846.22	1175	1.389	0.328
LS	CTTTCA	1172.91	1626	1.386	0.327
LS	TTAAGT	695.33	886	1.274	0.242
LS	CTCTCT	2104.05	2553	1.213	0.193
LS	CTAAGT	642.91	770	1.198	0.180
LS	CTCTCA	1701.00	2003	1.178	0.163
LS	CTTTCC	1667.81	1819	1.091	0.087
LS	TTGTCA	1149.26	1210	1.053	0.052
LS	CTGTCG	1329.18	1392	1.047	0.046
LS	TTGTCT	1421.58	1461	1.028	0.027
LS	CTGAGC	5694.68	5805	1.019	0.019
LS	CTGTCC	5026.41	4628	0.921	−0.083
LS	TTGAGT	1168.09	1035	0.886	−0.121
LS	TTGTCC	1634.18	1334	0.816	−0.203
LS	CTATCA	632.54	512	0.809	−0.211
LS	CTAAGC	1019.02	791	0.776	−0.253
LS	TTATCC	972.78	727	0.747	−0.291
LS	CTGAGT	3592.81	2665	0.742	−0.299
LS	CTTAGT	1192.13	856	0.718	−0.331
LS	CTATCT	782.42	557	0.712	−0.340
LS	CTGTCT	4372.48	2950	0.675	−0.394
LS	CTTTCG	441.04	291	0.660	−0.416
LS	TTGTCG	432.14	278	0.643	−0.441
LS	CTGTCA	3534.89	2228	0.630	−0.462
LS	TTGAGC	1851.45	1128	0.609	−0.496
LS	CTATCC	899.44	541	0.601	−0.508
LS	TTATCG	257.24	152	0.591	−0.526
LS	TTAAGC	1102.11	551	0.500	−0.693
LS	CTATCG	237.85	102	0.429	−0.847
LS	CTTAGC	1889.55	793	0.420	−0.868
LT	CTCACC	2534.19	4959	1.957	0.671
LT	CTCACG	832.47	1510	1.814	0.595
LT	TTAACA	825.09	1163	1.410	0.343
LT	CTCACT	1814.22	2521	1.390	0.329
LT	TTAACT	729.65	969	1.328	0.284
LT	CTAACT	674.64	817	1.211	0.191
LT	CTAACA	762.89	898	1.177	0.163
LT	CTCACA	2051.52	2374	1.157	0.146
LT	CTGACG	1729.98	1795	1.038	0.037
LT	TTGACT	1225.76	1259	1.027	0.027
LT	TTGACA	1386.09	1401	1.011	0.011
LT	CTTACT	1250.98	1259	1.006	0.006
LT	CTGACC	5266.36	5160	0.980	−0.020
LT	CTTACA	1414.61	1109	0.784	−0.243
LT	CTGACT	3770.17	2808	0.745	−0.295
LT	TTGACC	1712.20	1235	0.721	−0.327
LT	CTAACC	942.38	678	0.719	−0.329
LT	TTGACG	562.45	399	0.709	−0.343
LT	CTGACA	4263.32	3003	0.704	−0.350
LT	CTAACG	309.57	215	0.695	−0.365
LT	TTAACC	1019.22	687	0.674	−0.394
LT	CTTACC	1747.43	1104	0.632	−0.459
LT	TTAACG	334.81	164	0.490	−0.714
LT	CTTACG	574.02	247	0.430	−0.843
LV	CTTGTT	1029.60	1741	1.691	0.525
LV	TTAGTA	389.95	602	1.544	0.434
LV	TTGGTA	655.07	980	1.496	0.403
LV	CTTGTA	668.56	993	1.485	0.396
LV	CTGGTG	7859.41	11424	1.454	0.374
LV	CTAGTA	360.55	519	1.439	0.364
LV	TTGGTT	1008.84	1427	1.414	0.347
LV	CTTGTC	1318.22	1541	1.169	0.156
LV	TTAGTT	600.53	690	1.149	0.139
LV	CTGGTC	3972.81	4541	1.143	0.134
LV	TTGGTG	2555.25	2882	1.128	0.120
LV	CTAGTT	555.26	580	1.045	0.044
LV	TTGGTC	1291.64	1345	1.041	0.040
LV	CTTGTG	2607.83	2540	0.974	−0.026
LV	CTAGTG	1406.38	1272	0.904	−0.100
LV	CTGGTA	2014.87	1720	0.854	−0.158
LV	CTGGTT	3102.98	2576	0.830	−0.186
LV	CTAGTC	710.90	551	0.775	−0.255
LV	TTAGTG	1521.06	947	0.623	−0.474
LV	TTAGTC	768.87	416	0.541	−0.614
LV	CTCGTC	1911.73	1013	0.530	−0.635
LV	CTCGTG	3781.97	1691	0.447	−0.805
LV	CTCGTT	1493.16	373	0.250	−1.387
LV	CTCGTA	969.56	191	0.197	−1.625
LW	CTCTGG	1742.64	2796	1.604	0.473
LW	CTGTGG	3621.43	3365	0.929	−0.073
LW	CTTTGG	1201.63	1018	0.847	−0.166
LW	CTATGG	648.03	501	0.773	−0.257
LW	TTATGG	700.87	535	0.763	−0.270
LW	TTGTGG	1177.40	877	0.745	−0.295
LY	CTCTAC	2082.09	4204	2.019	0.703
LY	TTATAT	680.44	1022	1.502	0.407
LY	CTCTAT	1691.85	2487	1.470	0.385
LY	CTTTAT	1166.60	1591	1.364	0.310
LY	CTATAT	629.14	596	0.947	−0.054
LY	TTGTAT	1143.08	1063	0.930	−0.073
LY	CTGTAC	4326.84	3390	0.783	−0.244
LY	CTTTAC	1435.69	1069	0.745	−0.295
LY	TTGTAC	1406.74	1006	0.715	−0.335
LY	TTATAC	837.39	579	0.691	−0.369
LY	CTGTAT	3515.88	2202	0.626	−0.468
LY	CTATAC	774.26	481	0.621	−0.476
MA	ATGGCG	1645.46	2370	1.440	0.365
MA	ATGGCA	3503.58	3580	1.022	0.022
MA	ATGGCT	4002.27	4003	1.000	0.000
MA	ATGGCC	6085.70	5284	0.868	−0.141
MC	ATGTGT	1386.67	1448	1.044	0.043
MC	ATGTGC	1646.33	1585	0.963	−0.038
MD	ATGGAT	4467.48	4634	1.037	0.037
MD	ATGGAC	5046.52	4880	0.967	−0.034
ME	ATGGAG	8054.28	8223	1.021	0.021
ME	ATGGAA	6022.72	5854	0.972	−0.028
MF	ATGTTT	2565.53	2833	1.104	0.099
MF	ATGTTC	2936.47	2669	0.909	−0.096
MG	ATGGGC	3467.73	3533	1.019	0.019
MG	ATGGGT	1655.83	1675	1.012	0.012
MG	ATGGGA	2557.59	2526	0.988	−0.012
MG	ATGGGG	2496.85	2444	0.979	−0.021
MH	ATGCAT	1465.33	1478	1.009	0.009
MH	ATGCAC	2020.67	2008	0.994	−0.006
MI	ATGATT	2305.40	2382	1.033	0.033
MI	ATGATA	1060.28	1094	1.032	0.031
MI	ATGATC	2915.32	2805	0.962	−0.039
MK	ATGAAG	6107.32	6423	1.052	0.050
MK	ATGAAA	4715.68	4400	0.933	−0.069
ML	ATGCTG	5938.40	6536	1.101	0.096
ML	ATGCTA	1062.63	1122	1.056	0.054
ML	ATGTTG	1930.69	1922	0.995	−0.005
ML	ATGTTA	1149.28	1134	0.987	−0.013
ML	ATGCTT	1970.42	1887	0.958	−0.043
ML	ATGCTC	2857.58	2308	0.808	−0.214
MM	ATGATG	3925.00	3925	1.000	0.000
MN	ATGAAT	3249.30	3301	1.016	0.016
MN	ATGAAC	3578.70	3527	0.986	−0.015
MP	ATGCCC	2676.16	2752	1.028	0.028
MP	ATGCCA	2313.29	2313	1.000	0.000
MP	ATGCCT	2396.87	2372	0.990	−0.010
MP	ATGCCG	969.67	919	0.948	−0.054
MQ	ATGCAG	5141.70	5165	1.005	0.005
MQ	ATGCAA	1841.30	1818	0.987	−0.013
MR	ATGAGG	1626.37	2127	1.308	0.268
MR	ATGAGA	1656.63	1974	1.192	0.175
MR	ATGCGG	1642.64	1513	0.921	−0.082
MR	ATGCGT	643.02	531	0.826	−0.191
MR	ATGCGA	890.44	684	0.768	−0.264
MR	ATGCGC	1501.91	1132	0.754	−0.283
MS	ATGTCG	666.33	809	1.214	0.194
MS	ATGTCT	2191.95	2338	1.067	0.065
MS	ATGTCA	1772.07	1781	1.005	0.005
MS	ATGTCC	2519.77	2493	0.989	−0.011
MS	ATGAGT	1801.10	1770	0.983	−0.017
MS	ATGAGC	2854.78	2615	0.916	−0.088
MT	ATGACT	2098.83	2195	1.046	0.045
MT	ATGACC	2931.75	2927	0.998	−0.002
MT	ATGACA	2373.36	2337	0.985	−0.015
MT	ATGACG	963.07	908	0.943	−0.059
MV	ATGGTG	4813.46	5122	1.064	0.062
MV	ATGGTT	1900.41	1915	1.008	0.008
MV	ATGGTA	1234.00	1191	0.965	−0.035
MV	ATGGTC	2433.13	2153	0.885	−0.122
MW	ATGTGG	1876.00	1876	1.000	0.000
MY	ATGTAC	2354.66	2363	1.004	0.004
MY	ATGTAT	1913.34	1905	0.996	−0.004
NA	AATGCA	1705.68	3344	1.961	0.673
NA	AATGCT	1948.47	3458	1.775	0.574
NA	AATGCC	2962.77	4259	1.438	0.363
NA	AATGCG	801.08	624	0.779	−0.250
NA	AACGCG	882.29	661	0.749	−0.289
NA	AACGCC	3263.12	1899	0.582	−0.541
NA	AACGCA	1878.60	700	0.373	−0.987
NA	AACGCT	2146.00	643	0.300	−1.205
NC	AACTGC	1868.57	2826	1.512	0.414
NC	AACTGT	1573.86	2016	1.281	0.248
NC	AATTGT	1429.00	935	0.654	−0.424
NC	AATTGC	1696.57	791	0.466	−0.763
ND	AATGAT	2555.01	4420	1.730	0.548
ND	AATGAC	2886.18	4521	1.566	0.449
ND	AACGAC	3178.77	1654	0.520	−0.653
ND	AACGAT	2814.03	839	0.298	−1.210
NE	AATGAA	3381.19	7367	2.179	0.779
NE	AATGAG	4521.72	5796	1.282	0.248
NE	AACGAG	4980.12	2476	0.497	−0.699
NE	AACGAA	3723.97	968	0.260	−1.347
NF	AACTTC	3150.86	4259	1.352	0.301
NF	AACTTT	2752.85	2846	1.034	0.033
NF	AATTTT	2499.46	2350	0.940	−0.062
NF	AATTTC	2860.84	1809	0.632	−0.458
NG	AATGGA	2235.93	4484	2.005	0.696
NG	AATGGT	1447.59	2430	1.679	0.518
NG	AATGGG	2182.83	3202	1.467	0.383
NG	AATGGC	3031.62	4001	1.320	0.277
NG	AACGGG	2404.12	1508	0.627	−0.466
NG	AACGGC	3338.95	1752	0.525	−0.645
NG	AACGGA	2462.61	804	0.326	−1.119
NG	AACGGT	1594.34	517	0.324	−1.126
NH	AACCAC	2167.68	2776	1.281	0.247
NH	AACCAT	1571.93	1639	1.043	0.042
NH	AATCAT	1427.24	1456	1.020	0.020
NH	AATCAC	1968.15	1264	0.642	−0.443
NI	AACATC	3876.27	5487	1.416	0.348
NI	AACATT	3065.31	3184	1.039	0.038
NI	AATATA	1280.01	1309	1.023	0.022
NI	AACATA	1409.77	1384	0.982	−0.018
NI	AATATT	2783.16	2725	0.979	−0.021
NI	AATATC	3519.48	1845	0.524	−0.646
NK	AACAAG	4824.98	5918	1.227	0.204
NK	AACAAA	3725.54	4221	1.133	0.125
NK	AATAAA	3382.62	3607	1.066	0.064
NK	AATAAG	4380.86	2568	0.586	−0.534
NL	AATTTA	1025.31	1571	1.532	0.427
NL	AACCTC	2807.78	3954	1.408	0.342
NL	AACTTG	1897.05	2429	1.280	0.247
NL	AACCTG	5834.92	6690	1.147	0.137
NL	AATTTG	1722.43	1947	1.130	0.123
NL	AATCTT	1757.88	1943	1.105	0.100
NL	AACCTA	1044.12	1135	1.087	0.083
NL	AACCTT	1936.08	2021	1.044	0.043
NL	AACTTA	1129.25	1129	1.000	0.000
NL	AATCTA	948.01	893	0.942	−0.060
NL	AATCTC	2549.34	1713	0.672	−0.398
NL	AATCTG	5297.84	2525	0.477	−0.741
NM	AACATG	3351.76	4374	1.305	0.266
NM	AATATG	3043.24	2021	0.664	−0.409
NN	AACAAC	3150.02	4430	1.406	0.341
NN	AACAAT	2860.08	2830	0.989	−0.011
NN	AATAAT	2596.82	2424	0.933	−0.069
NN	AATAAC	2860.08	1783	0.623	−0.473
NP	AACCCC	2770.02	3474	1.254	0.226
NP	AATCCA	2174.02	2380	1.095	0.091
NP	AACCCA	2394.42	2612	1.091	0.087
NP	AATCCT	2252.58	2414	1.072	0.069
NP	AACCCG	1003.68	1048	1.044	0.043
NP	AACCCT	2480.94	2578	1.039	0.038
NP	AATCCC	2515.05	1641	0.652	−0.427
NP	AATCCG	911.29	355	0.390	−0.943
NQ	AATCAA	1516.57	1905	1.256	0.228
NQ	AACCAA	1670.31	1955	1.170	0.157
NQ	AACCAG	4664.22	5409	1.160	0.148
NQ	AATCAG	4234.90	2817	0.665	−0.408
NR	AACAGA	1511.98	2383	1.576	0.455
NR	AACCGC	1370.77	1966	1.434	0.361
NR	AACAGG	1484.36	1903	1.282	0.248
NR	AACCGA	812.69	998	1.228	0.205
NR	AACCGT	586.88	706	1.203	0.185
NR	AACCGG	1499.21	1779	1.187	0.171
NR	AATCGA	737.89	687	0.931	−0.071
NR	AATCGT	532.86	486	0.912	−0.092
NR	AATAGA	1372.81	1117	0.814	−0.206
NR	AATCGC	1244.60	602	0.484	−0.726
NR	AATAGG	1347.73	643	0.477	−0.740
NR	AATCGG	1361.22	593	0.436	−0.831
NS	AACAGC	2917.73	4490	1.539	0.431
NS	AACAGT	1840.81	2414	1.311	0.271
NS	AACTCG	681.02	821	1.206	0.187
NS	AATTCA	1644.43	1970	1.198	0.181
NS	AATTCT	2034.08	2383	1.172	0.158
NS	AACTCC	2575.33	2818	1.094	0.090
NS	AACTCA	1811.14	1783	0.984	−0.016
NS	AACTCT	2240.29	1981	0.884	−0.123
NS	AATAGT	1671.38	1193	0.714	−0.337
NS	AATTCC	2338.29	1655	0.708	−0.346
NS	AATAGC	2649.17	1273	0.481	−0.733
NS	AATTCG	618.33	241	0.390	−0.942
NT	AACACG	860.22	1238	1.439	0.364
NT	AACACA	2119.90	2783	1.313	0.272
NT	AACACC	2618.65	3278	1.252	0.225
NT	AACACT	1874.68	2099	1.120	0.113
NT	AATACT	1702.13	1540	0.905	−0.100
NT	AATACA	1924.77	1692	0.879	−0.129
NT	AATACC	2377.62	1312	0.552	−0.595
NT	AATACG	781.04	317	0.406	−0.902
NV	AATGTA	927.15	1710	1.844	0.612
NV	AATGTT	1427.85	2573	1.802	0.589
NV	AATGTC	1828.10	2877	1.574	0.453
NV	AATGTG	3616.54	4314	1.193	0.176
NV	AACGTG	3983.18	2772	0.696	−0.363
NV	AACGTC	2013.43	1341	0.666	−0.406
NV	AACGTT	1572.60	509	0.324	−1.128
NV	AACGTA	1021.14	294	0.288	−1.245
NW	AACTGG	1808.22	2595	1.435	0.361
NW	AATTGG	1641.78	855	0.521	−0.652
NY	AACTAC	2506.72	3191	1.273	0.241
NY	AACTAT	2036.89	2145	1.053	0.052
NY	AATTAT	1849.41	1795	0.971	−0.030
NY	AATTAC	2275.98	1538	0.676	−0.392
PA	CCGGCG	470.57	1166	2.478	0.907
PA	CCGGCC	1740.39	2666	1.532	0.426
PA	CCAGCA	2390.31	3368	1.409	0.343
PA	CCAGCT	2730.54	3622	1.326	0.283
PA	CCTGCT	2829.20	3750	1.325	0.282
PA	CCTGCA	2476.67	3178	1.283	0.249
PA	CCAGCC	4151.96	4942	1.190	0.174
PA	CCCGCG	1298.71	1528	1.177	0.163
PA	CCTGCC	4301.98	5000	1.162	0.150
PA	CCAGCG	1122.61	1078	0.960	−0.041
PA	CCTGCG	1163.17	1105	0.950	−0.051
PA	CCGGCT	1144.57	1013	0.885	−0.122
PA	CCGGCA	1001.95	777	0.775	−0.254
PA	CCCGCC	4803.25	2690	0.560	−0.580
PA	CCCGCA	2765.26	846	0.306	−1.184
PA	CCCGCT	3158.86	821	0.260	−1.347
PC	CCCTGC	1550.51	2870	1.851	0.616
PC	CCCTGT	1305.97	1577	1.208	0.189
PC	CCGTGC	561.80	630	1.121	0.115
PC	CCTTGT	1169.67	1001	0.856	−0.156
PC	CCATGT	1128.89	831	0.736	−0.306
PC	CCGTGT	473.20	340	0.719	−0.331
PC	CCTTGC	1388.69	937	0.675	−0.393
PC	CCATGC	1340.27	733	0.547	−0.603
PD	CCAGAT	2721.60	4165	1.530	0.425
PD	CCTGAT	2819.94	3781	1.341	0.293
PD	CCGGAC	1288.69	1659	1.287	0.253
PD	CCAGAC	3074.36	3766	1.225	0.203
PD	CCTGAC	3185.44	3646	1.145	0.135
PD	CCGGAT	1140.82	895	0.785	−0.243
PD	CCCGAC	3556.62	2215	0.623	−0.474
PD	CCCGAT	3148.53	809	0.257	−1.359
PE	CCAGAA	3999.86	5699	1.425	0.354
PE	CCTGAG	5542.36	7122	1.285	0.251
PE	CCGGAG	2242.20	2870	1.280	0.247
PE	CCAGAG	5349.08	6777	1.267	0.237
PE	CCTGAA	4144.39	5108	1.233	0.209
PE	CCCGAG	6188.17	4149	0.670	−0.400
PE	CCGGAA	1676.64	1032	0.616	−0.485
PE	CCCGAA	4627.30	1013	0.219	−1.519
PF	CCCTTC	2555.92	4301	1.683	0.520
PF	CCATTT	1930.27	2057	1.066	0.064
PF	CCTTTT	2000.01	1967	0.983	−0.017
PF	CCCTTT	2233.06	2159	0.967	−0.034
PF	CCTTTC	2289.18	2078	0.908	−0.097
PF	CCGTTC	926.10	662	0.715	−0.336
PF	CCATTC	2209.35	1290	0.584	−0.538
PF	CCGTTT	809.12	439	0.543	−0.611
PG	CCTGGG	2918.52	4310	1.477	0.390
PG	CCTGGA	2989.52	4317	1.444	0.367
PG	CCGGGC	1639.82	2353	1.435	0.361
PG	CCGGGG	1180.71	1657	1.403	0.339
PG	CCTGGT	1935.48	2673	1.381	0.323
PG	CCAGGA	2885.27	3897	1.351	0.301
PG	CCAGGG	2816.75	3472	1.233	0.209
PG	CCAGGT	1867.98	2259	1.209	0.190
PG	CCTGGC	4053.37	4622	1.140	0.131
PG	CCAGGC	3912.02	4106	1.050	0.048
PG	CCGGGT	783.01	661	0.844	−0.169
PG	CCGGGA	1209.43	963	0.796	−0.228
PG	CCCGGG	3258.60	2136	0.655	−0.422
PG	CCCGGC	4525.68	2555	0.565	−0.572
PG	CCCGGA	3337.86	968	0.290	−1.238
PG	CCCGGT	2161.00	526	0.243	−1.413
PH	CCGCAC	725.13	972	1.340	0.293
PH	CCCCAC	2001.25	2505	1.252	0.225
PH	CCTCAT	1299.79	1592	1.225	0.203
PH	CCACAT	1254.46	1222	0.974	−0.026
PH	CCCCAT	1451.24	1303	0.898	−0.108
PH	CCTCAC	1792.40	1531	0.854	−0.158
PH	CCACAC	1729.89	1366	0.790	−0.236
PH	CCGCAT	525.84	289	0.550	−0.599
PI	CCCATC	2119.04	4651	2.195	0.786
PI	CCCATT	1675.71	2102	1.254	0.227
PI	CCAATA	666.18	819	1.229	0.207
PI	CCCATA	770.68	776	1.007	0.007
PI	CCAATT	1448.49	1386	0.957	−0.044
PI	CCTATA	690.25	603	0.874	−0.135
PI	CCTATT	1500.83	1266	0.844	−0.170
PI	CCAATC	1831.71	939	0.513	−0.668
PI	CCTATC	1897.89	957	0.504	−0.685
PI	CCGATT	607.17	299	0.492	−0.708
PI	CCGATC	767.80	342	0.445	−0.809
PI	CCGATA	279.24	115	0.412	−0.887
PK	CCCAAG	3738.47	6383	1.707	0.535
PK	CCCAAA	2886.60	3787	1.312	0.271
PK	CCAAAA	2495.20	2489	0.998	−0.002
PK	CCAAAG	3231.55	3127	0.968	−0.033
PK	CCTAAA	2585.35	1840	0.712	−0.340
PK	CCGAAG	1354.58	940	0.694	−0.365
PK	CCTAAG	3348.32	1660	0.496	−0.702
PK	CCGAAA	1045.92	460	0.440	−0.821
PL	CCGCTG	1824.84	3343	1.832	0.605
PL	CCGCTC	878.12	1254	1.428	0.356
PL	CCTTTG	1466.52	2054	1.401	0.337
PL	CCTTTA	872.97	1195	1.369	0.314
PL	CCCTTG	1637.40	2122	1.296	0.259
PL	CCTCTT	1496.70	1827	1.221	0.199
PL	CCCCTG	5036.31	5760	1.144	0.134
PL	CCCCTC	2423.49	2646	1.092	0.088
PL	CCTCTA	807.16	871	1.079	0.076
PL	CCATTA	842.53	826	0.980	−0.020
PL	CCACTT	1444.51	1371	0.949	−0.052
PL	CCACTA	779.01	729	0.936	−0.066
PL	CCTCTC	2170.57	1934	0.891	−0.115
PL	CCTCTG	4510.71	3745	0.830	−0.186
PL	CCATTG	1415.38	1172	0.828	−0.189
PL	CCCCTT	1671.10	1324	0.792	−0.233
PL	CCGCTA	326.54	255	0.781	−0.247
PL	CCCCTA	901.21	689	0.765	−0.268
PL	CCACTG	4353.41	3218	0.739	−0.302
PL	CCCTTA	974.69	709	0.727	−0.318
PL	CCACTC	2094.88	1475	0.704	−0.351
PL	CCGTTG	593.29	402	0.678	−0.389
PL	CCGCTT	605.50	402	0.664	−0.410
PL	CCGTTA	353.17	157	0.445	−0.811
PM	CCCATG	2307.54	3923	1.700	0.531
PM	CCAATG	1994.65	1552	0.778	−0.251
PM	CCGATG	836.10	520	0.622	−0.475
PM	CCTATG	2066.72	1210	0.585	−0.535
PN	CCCAAC	2313.61	4255	1.839	0.609
PN	CCAAAT	1815.81	2453	1.351	0.301
PN	CCCAAT	2100.65	2296	1.093	0.089
PN	CCAAAC	1999.90	1735	0.868	−0.142
PN	CCTAAT	1881.42	1342	0.713	−0.338
PN	CCTAAC	2072.16	997	0.481	−0.732
PN	CCGAAT	761.14	340	0.447	−0.806
PP	CCGCCG	608.57	2335	3.837	1.345
PP	CCGCCC	1679.58	2697	1.606	0.474
PP	CCCCCG	1679.58	2420	1.441	0.365
PP	CCTCCA	3588.72	4314	1.202	0.184
PP	CCTCCT	3718.39	4305	1.158	0.146
PP	CCACCA	3463.58	3850	1.112	0.106
PP	CCACCT	3588.72	3798	1.058	0.057
PP	CCCCCA	4006.89	4095	1.022	0.022
PP	CCACCC	4006.89	3595	0.897	−0.108
PP	CCGCCA	1451.84	1280	0.882	−0.126
PP	CCACCG	1451.84	1252	0.862	−0.148
PP	CCGCCT	1504.30	1286	0.855	−0.157
PP	CCTCCC	4151.67	3338	0.804	−0.218
PP	CCTCCG	1504.30	1152	0.766	−0.267
PP	CCCCCT	4151.67	3160	0.761	−0.273
PP	CCCCCC	4635.43	2315	0.499	−0.694
PQ	CCCCAG	5063.98	6421	1.268	0.237
PQ	CCGCAG	1834.86	2187	1.192	0.176
PQ	CCTCAA	1624.21	1752	1.079	0.076
PQ	CCTCAG	4535.49	4221	0.931	−0.072
PQ	CCACAA	1567.57	1405	0.896	−0.109
PQ	CCACAG	4377.33	3670	0.838	−0.176
PQ	CCCCAA	1813.47	1497	0.825	−0.192
PQ	CCGCAA	657.08	321	0.489	−0.716
PR	CCGCGC	563.43	1094	1.942	0.664
PR	CCGCGG	616.23	1113	1.806	0.591
PR	CCCAGG	1683.86	2927	1.738	0.553
PR	CCCCGG	1700.71	2608	1.533	0.428
PR	CCCCGC	1555.00	1979	1.273	0.241
PR	CCCCGA	921.92	1166	1.265	0.235
PR	CCTCGA	825.71	1015	1.229	0.206
PR	CCAAGA	1482.62	1608	1.085	0.081
PR	CCTCGT	596.27	644	1.080	0.077
PR	CCCAGA	1715.19	1801	1.050	0.049
PR	CCGAGG	610.12	636	1.042	0.042
PR	CCTCGG	1523.22	1511	0.992	−0.008
PR	CCCCGT	665.75	655	0.984	−0.016
PR	CCAAGG	1455.54	1347	0.925	−0.077
PR	CCACGA	796.91	632	0.793	−0.232
PR	CCGCGT	241.23	191	0.792	−0.233
PR	CCACGT	575.48	418	0.726	−0.320
PR	CCACGG	1470.10	1040	0.707	−0.346
PR	CCGCGA	334.04	226	0.677	−0.391
PR	CCTCGC	1392.72	838	0.602	−0.508
PR	CCACGC	1344.15	701	0.522	−0.651
PR	CCGAGA	621.48	308	0.496	−0.702
PR	CCTAGA	1536.19	692	0.450	−0.797
PR	CCTAGG	1508.13	586	0.389	−0.945
PS	CCCAGC	3196.25	6398	2.002	0.694
PS	CCCTCG	746.03	1385	1.856	0.619
PS	CCGTCG	270.31	483	1.787	0.580
PS	CCCAGT	2016.53	2743	1.360	0.308
PS	CCTTCA	1776.97	2263	1.274	0.242
PS	CCTTCT	2198.02	2711	1.233	0.210
PS	CCCTCC	2821.16	3353	1.189	0.173
PS	CCATCA	1715.00	1819	1.061	0.059
PS	CCATCT	2121.37	2183	1.029	0.029
PS	CCTTCC	2526.74	2594	1.027	0.026
PS	CCGTCC	1022.21	1048	1.025	0.025
PS	CCCTCA	1984.02	1945	0.980	−0.020
PS	CCAAGT	1743.10	1582	0.908	−0.097
PS	CCCTCT	2454.14	2113	0.861	−0.150
PS	CCTTCG	668.17	552	0.826	−0.191
PS	CCATCC	2438.63	1995	0.818	−0.201
PS	CCGAGC	1158.11	885	0.764	−0.269
PS	CCATCG	644.87	475	0.737	−0.306
PS	CCAAGC	2762.85	1659	0.600	−0.510
PS	CCGTCT	889.22	523	0.588	−0.531
PS	CCGAGT	730.66	371	0.508	−0.678
PS	CCGTCA	718.88	364	0.506	−0.681
PS	CCTAGT	1806.08	860	0.476	−0.742
PS	CCTAGC	2862.68	968	0.338	−1.084
PT	CCCACG	829.55	1764	2.126	0.754
PT	CCCACC	2525.29	4586	1.816	0.597
PT	CCCACA	2044.32	2719	1.330	0.285
PT	CCCACT	1807.85	2282	1.262	0.233
PT	CCAACA	1767.12	1895	1.072	0.070
PT	CCAACT	1562.71	1593	1.019	0.019
PT	CCGACG	300.57	305	1.015	0.015
PT	CCTACT	1619.18	1252	0.773	−0.257
PT	CCAACC	2182.87	1514	0.694	−0.366
PT	CCTACA	1830.97	1241	0.678	−0.389
PT	CCGACC	915.00	592	0.647	−0.435
PT	CCAACG	717.06	463	0.646	−0.437
PT	CCTACC	2261.75	1251	0.553	−0.592
PT	CCGACT	655.05	342	0.522	−0.650
PT	CCGACA	740.73	352	0.475	−0.744
PT	CCTACG	742.97	352	0.474	−0.747
PV	CCTGTT	1493.79	2375	1.590	0.464
PV	CCTGTA	969.97	1482	1.528	0.424
PV	CCAGTA	936.15	1352	1.444	0.368
PV	CCTGTG	3783.57	5362	1.417	0.349
PV	CCAGTT	1441.70	2038	1.414	0.346
PV	CCTGTC	1912.53	2666	1.394	0.332
PV	CCGGTG	1530.67	1911	1.248	0.222
PV	CCAGTG	3651.63	3787	1.037	0.036
PV	CCAGTC	1845.84	1863	1.009	0.009
PV	CCGGTC	773.73	778	1.006	0.006
PV	CCCGTG	4224.44	2576	0.610	−0.495
PV	CCGGTT	604.32	351	0.581	−0.543
PV	CCGGTA	392.41	215	0.548	−0.602
PV	CCCGTC	2135.39	1084	0.508	−0.678
PV	CCCGTT	1667.85	391	0.234	−1.451
PV	CCCGTA	1083.00	216	0.199	−1.612
PW	CCCTGG	1769.80	2753	1.556	0.442
PW	CCGTGG	641.26	661	1.031	0.030
PW	CCATGG	1529.83	1060	0.693	−0.367
PW	CCTTGG	1585.10	1052	0.664	−0.410
PY	CCCTAC	2166.25	3378	1.559	0.444
PY	CCCTAT	1760.24	2097	1.191	0.175
PY	CCTTAT	1576.54	1702	1.080	0.077
PY	CCATAT	1521.56	1513	0.994	−0.006
PY	CCTTAC	1940.18	1485	0.765	−0.267
PY	CCGTAC	784.91	592	0.754	−0.282
PY	CCGTAT	637.80	429	0.673	−0.397
PY	CCATAC	1872.52	1064	0.568	−0.565
QA	CAAGCA	1597.87	2339	1.464	0.381
QA	CAAGCT	1825.31	2409	1.320	0.277
QA	CAGGCG	2095.55	2271	1.084	0.080
QA	CAGGCC	7750.37	7695	0.993	−0.007
QA	CAAGCC	2775.49	2655	0.957	−0.044
QA	CAGGCT	5097.04	4584	0.899	−0.106
QA	CAGGCA	4461.94	3943	0.884	−0.124
QA	CAAGCG	750.44	458	0.610	−0.494
QC	CAGTGT	2490.13	2791	1.121	0.114
QC	CAGTGC	2956.40	3260	1.103	0.098
QC	CAATGT	891.74	822	0.922	−0.081
QC	CAATGC	1058.72	524	0.495	−0.703
QD	CAAGAT	2128.42	3326	1.563	0.446
QD	CAAGAC	2404.29	2506	1.042	0.041
QD	CAGGAC	6713.82	6642	0.989	−0.011
QD	CAGGAT	5943.46	4716	0.793	−0.231
QE	CAAGAA	3247.03	5286	1.628	0.487
QE	CAGGAG	12125.58	12556	1.035	0.035
QE	CAAGAG	4342.30	4206	0.969	−0.032
QE	CAGGAA	9067.09	6734	0.743	−0.297
QF	CAGTTT	3509.26	4032	1.149	0.139
QF	CAGTTC	4016.64	4205	1.047	0.046
QF	CAATTT	1256.70	1156	0.920	−0.084
QF	CAATTC	1438.40	828	0.576	−0.552
QG	CAAGGA	1440.03	2837	1.970	0.678
QG	CAAGGT	932.30	1506	1.615	0.480
QG	CAAGGG	1405.83	1700	1.209	0.190
QG	CAAGGC	1952.47	2192	1.123	0.116
QG	CAGGGC	5452.14	5605	1.028	0.028
QG	CAGGGT	2603.39	2292	0.880	−0.127
QG	CAGGGA	4021.17	2871	0.714	−0.337
QG	CAGGGG	3925.67	2730	0.695	−0.363
QH	CAACAT	1067.82	1364	1.277	0.245
QH	CAGCAC	4111.88	4483	1.090	0.086
QH	CAGCAT	2981.80	2794	0.937	−0.065
QH	CAACAC	1472.51	993	0.674	−0.394
QI	CAAATA	656.37	1125	1.714	0.539
QI	CAAATT	1427.17	1667	1.168	0.155
QI	CAGATC	5039.60	5197	1.031	0.031
QI	CAGATA	1832.87	1802	0.983	−0.017
QI	CAGATT	3985.26	3693	0.927	−0.076
QI	CAAATC	1804.74	1262	0.699	−0.358
QK	CAGAAG	8990.94	9726	1.082	0.079
QK	CAAAAA	2486.09	2610	1.050	0.049
QK	CAGAAA	6942.22	6532	0.941	−0.061
QK	CAAAAG	3219.76	2771	0.861	−0.150
QL	CAGCTG	10304.18	12629	1.226	0.203
QL	CAACTA	660.31	798	1.209	0.189
QL	CAACTT	1224.39	1479	1.208	0.189
QL	CAGCTC	4958.40	5986	1.207	0.188
QL	CAGCTA	1843.86	2002	1.086	0.082
QL	CAGCTT	3419.03	3476	1.017	0.017
QL	CAATTA	714.15	642	0.899	−0.107
QL	CAGTTG	3350.09	2597	0.775	−0.255
QL	CAGTTA	1994.20	1518	0.761	−0.273
QL	CAACTC	1775.66	1279	0.720	−0.328
QL	CAACTG	3690.04	2093	0.567	−0.567
QL	CAATTG	1199.70	635	0.529	−0.636
QM	CAGATG	5587.91	5592	1.001	0.001
QM	CAAATG	2001.09	1997	0.998	−0.002
QN	CAAAAT	1720.47	2394	1.391	0.330
QN	CAGAAC	5291.34	5195	0.982	−0.018
QN	CAGAAT	4804.30	4430	0.922	−0.081
QN	CAAAAC	1894.89	1692	0.893	−0.113
QP	CAGCCG	1816.66	2237	1.231	0.208
QP	CAGCCC	5013.75	6143	1.225	0.203
QP	CAGCCT	4490.51	4526	1.008	0.008
QP	CAGCCA	4333.91	4235	0.977	−0.023
QP	CAACCA	1552.02	1441	0.928	−0.074
QP	CAACCT	1608.10	1304	0.811	−0.210
QP	CAACCC	1795.48	1132	0.630	−0.461
QP	CAACCG	650.57	243	0.374	−0.985
QQ	CAACAA	1545.49	1866	1.207	0.188
QQ	CAGCAG	12051.19	13131	1.090	0.086
QQ	CAGCAA	4315.66	4034	0.935	−0.067
QQ	CAACAG	4315.66	3197	0.741	−0.300
QR	CAAAGA	1214.45	1863	1.534	0.428
QR	CAGAGG	3329.32	4331	1.301	0.263
QR	CAAAGG	1192.27	1360	1.141	0.132
QR	CAGAGA	3391.27	3777	1.114	0.108
QR	CAGCGC	3074.54	3169	1.031	0.030
QR	CAGCGG	3362.63	3352	0.997	−0.003
QR	CAGCGT	1316.32	1215	0.923	−0.080
QR	CAGCGA	1822.82	1469	0.806	−0.216
QR	CAACGT	471.39	327	0.694	−0.366
QR	CAACGA	652.77	413	0.633	−0.458
QR	CAACGG	1204.20	453	0.376	−0.978
QR	CAACGC	1101.03	404	0.367	−1.003
QS	CAAAGT	904.91	1408	1.556	0.442
QS	CAGAGC	4005.17	5248	1.310	0.270
QS	CAGAGT	2526.89	2963	1.173	0.159
QS	CAAAGC	1434.30	1465	1.021	0.021
QS	CAGTCG	934.84	923	0.987	−0.013
QS	CAGTCA	2486.15	2379	0.957	−0.044
QS	CAGTCT	3075.24	2806	0.912	−0.092
QS	CAATCA	890.32	781	0.877	−0.131
QS	CAGTCC	3535.16	3051	0.863	−0.147
QS	CAATCT	1101.28	765	0.695	−0.364
QS	CAATCC	1265.98	587	0.464	−0.769
QS	CAATCG	334.78	119	0.355	−1.034
QT	CAAACT	1116.05	1463	1.311	0.271
QT	CAAACA	1262.03	1602	1.269	0.239
QT	CAGACG	1430.02	1665	1.164	0.152
QT	CAGACC	4353.25	4301	0.988	−0.012
QT	CAGACA	3524.12	3445	0.978	−0.023
QT	CAGACT	3116.48	2792	0.896	−0.110
QT	CAAACC	1558.95	1232	0.790	−0.235
QT	CAAACG	512.11	373	0.728	−0.317
QV	CAAGTA	657.01	1210	1.842	0.611
QV	CAAGTT	1011.82	1737	1.717	0.540
QV	CAAGTC	1295.45	1468	1.133	0.125
QV	CAAGTG	2562.79	2712	1.058	0.057
QV	CAGGTG	7156.41	7062	0.987	−0.013
QV	CAGGTC	3617.45	3213	0.888	−0.119
QV	CAGGTT	2825.43	2269	0.803	−0.219
QV	CAGGTA	1834.65	1290	0.703	−0.352
QW	CAGTGG	3057.92	3447	1.127	0.120
QW	CAATGG	1095.08	706	0.645	−0.439
QY	CAATAT	1029.01	1120	1.088	0.085
QY	CAGTAC	3536.21	3820	1.080	0.077
QY	CAGTAT	2873.43	2979	1.037	0.036
QY	CAATAC	1266.36	786	0.621	−0.477
RA	CGGGCG	659.18	1185	1.798	0.587
RA	CGGGCC	2437.97	3513	1.441	0.365
RA	AGAGCA	1415.51	1970	1.392	0.331
RA	CGCGCG	602.71	827	1.372	0.316
RA	CGTGCC	954.35	1266	1.327	0.283
RA	CGAGCA	760.84	970	1.275	0.243
RA	CGAGCT	869.13	1108	1.275	0.243
RA	CGAGCC	1321.57	1595	1.207	0.188
RA	AGAGCT	1616.99	1949	1.205	0.187
RA	CGTGCT	627.63	744	1.185	0.170
RA	CGGGCA	1403.55	1612	1.149	0.138
RA	CGTGCA	549.43	570	1.037	0.037
RA	CGTGCG	258.04	250	0.969	−0.032
RA	CGAGCG	357.33	341	0.954	−0.047
RA	AGGGCC	2413.81	2173	0.900	−0.105
RA	AGAGCC	2458.73	2202	0.896	−0.110
RA	CGGGCT	1603.33	1435	0.895	−0.111
RA	AGGGCA	1389.65	1242	0.894	−0.112
RA	AGGGCT	1587.45	1311	0.826	−0.191
RA	AGGGCG	652.65	524	0.803	−0.220
RA	CGCGCC	2229.09	1712	0.768	−0.264
RA	AGAGCG	664.79	384	0.578	−0.549
RA	CGCGCA	1283.30	331	0.258	−1.355
RA	CGCGCT	1465.97	369	0.252	−1.379
RC	CGCTGC	986.26	2873	2.913	1.069
RC	CGCTGT	830.71	1313	1.581	0.458
RC	CGTTGT	355.66	320	0.900	−0.106
RC	CGTTGC	422.25	372	0.881	−0.127
RC	AGATGT	916.29	806	0.880	−0.128
RC	CGATGT	492.51	421	0.855	−0.157
RC	AGGTGT	899.55	671	0.746	−0.293
RC	AGGTGC	1067.99	758	0.710	−0.343
RC	CGATGC	584.73	381	0.652	−0.428
RC	CGGTGC	1078.67	660	0.612	−0.491
RC	AGATGC	1087.86	642	0.590	−0.527
RC	CGGTGT	908.55	414	0.456	−0.786
RD	AGAGAT	2027.66	2952	1.456	0.376
RD	CGGGAC	2271.13	3231	1.423	0.353
RD	CGAGAT	1089.87	1500	1.376	0.319
RD	CGAGAC	1231.14	1693	1.375	0.319
RD	CGTGAC	889.05	1044	1.174	0.161
RD	AGAGAC	2290.48	2433	1.062	0.060
RD	CGTGAT	787.04	833	1.058	0.057
RD	AGGGAC	2248.63	2322	1.033	0.032
RD	AGGGAT	1990.62	1732	0.870	−0.139
RD	CGGGAT	2010.54	1606	0.799	−0.225
RD	CGCGAC	2076.56	1092	0.526	−0.643
RD	CGCGAT	1838.29	313	0.170	−1.770
RE	AGAGAA	2644.21	4195	1.586	0.462
RE	CGGGAG	3506.29	5344	1.524	0.421
RE	CGAGAG	1900.69	2475	1.302	0.264
RE	CGAGAA	1421.27	1844	1.297	0.260
RE	CGTGAG	1372.55	1453	1.059	0.057
RE	AGGGAG	3471.55	3469	0.999	−0.001
RE	AGAGAG	3536.15	3392	0.959	−0.042
RE	CGTGAA	1026.35	947	0.923	−0.080
RE	AGGGAA	2595.91	2343	0.903	−0.103
RE	CGGGAA	2621.88	2131	0.813	−0.207
RE	CGCGAG	3205.89	1839	0.574	−0.556
RE	CGCGAA	2397.25	268	0.112	−2.191
RF	CGCTTC	1446.49	3411	2.358	0.858
RF	CGTTTC	619.29	823	1.329	0.284
RF	CGTTTT	541.07	705	1.303	0.265
RF	AGATTT	1393.96	1531	1.098	0.094
RF	CGCTTT	1263.77	1366	1.081	0.078
RF	CGATTT	749.26	772	1.030	0.030
RF	AGGTTT	1368.50	1295	0.946	−0.055
RF	AGGTTC	1566.36	1192	0.761	−0.273
RF	CGATTC	857.59	632	0.737	−0.305
RF	CGGTTC	1582.03	951	0.601	−0.509
RF	AGATTC	1595.50	944	0.592	−0.525
RF	CGGTTT	1382.19	744	0.538	−0.619
RG	CGTGGT	370.38	685	1.849	0.615
RG	CGTGGG	558.50	980	1.755	0.562
RG	CGTGGC	775.66	1315	1.695	0.528
RG	CGAGGA	792.21	1266	1.598	0.469
RG	CGAGGG	773.39	1219	1.576	0.455
RG	AGAGGA	1473.87	2281	1.548	0.437
RG	CGAGGT	512.89	789	1.538	0.431
RG	CGGGGC	1981.48	2952	1.490	0.399
RG	CGTGGA	572.08	844	1.475	0.389
RG	CGAGGC	1074.12	1569	1.461	0.379
RG	AGAGGT	954.21	1128	1.182	0.167
RG	CGGGGT	946.15	918	0.970	−0.030
RG	CGCGGC	1811.72	1574	0.869	−0.141
RG	AGGGGC	1961.86	1660	0.846	−0.167
RG	AGAGGC	1998.36	1680	0.841	−0.174
RG	AGAGGG	1438.87	1203	0.836	−0.179
RG	AGGGGT	936.78	777	0.829	−0.187
RG	CGGGGG	1426.72	1146	0.803	−0.219
RG	CGGGGA	1461.42	1140	0.780	−0.248
RG	CGCGGG	1304.48	904	0.693	−0.367
RG	AGGGGA	1446.94	923	0.638	−0.450
RG	AGGGGG	1412.58	683	0.484	−0.727
RG	CGCGGT	865.09	248	0.287	−1.249
RG	CGCGGA	1336.22	302	0.226	−1.487
RH	CGCCAC	1288.00	1861	1.445	0.368
RH	CGGCAC	1408.69	1707	1.212	0.192
RH	AGACAT	1030.24	1201	1.166	0.153
RH	CGTCAT	399.89	447	1.118	0.111
RH	AGGCAT	1011.41	988	0.977	−0.023
RH	CGACAT	553.75	530	0.957	−0.044
RH	AGGCAC	1394.73	1292	0.926	−0.077
RH	AGACAC	1420.69	1212	0.853	−0.159
RH	CGTCAC	551.44	468	0.849	−0.164
RH	CGACAC	763.62	614	0.804	−0.218
RH	CGCCAT	934.02	728	0.779	−0.249
RH	CGGCAT	1021.53	730	0.715	−0.336
RI	CGCATC	1625.56	2948	1.814	0.595
RI	AGAATA	652.11	1175	1.802	0.589
RI	AGAATT	1417.90	2185	1.541	0.432
RI	AGGATA	640.20	804	1.256	0.228
RI	CGAATA	350.51	439	1.252	0.225
RI	CGAATT	762.13	850	1.115	0.109
RI	AGGATT	1392.00	1366	0.981	−0.019
RI	AGGATC	1760.27	1662	0.944	−0.057
RI	CGAATC	963.75	802	0.832	−0.184
RI	CGGATC	1777.88	1479	0.832	−0.184
RI	AGAATC	1793.03	1389	0.775	−0.255
RI	CGTATT	550.36	408	0.741	−0.299
RI	CGCATT	1285.48	913	0.710	−0.342
RI	CGGATA	646.60	451	0.697	−0.360
RI	CGTATC	695.96	440	0.632	−0.459
RI	CGTATA	253.12	152	0.601	−0.510
RI	CGGATT	1405.93	825	0.587	−0.533
RI	CGCATA	591.21	276	0.467	−0.762
RK	AGGAAG	3199.71	4856	1.518	0.417
RK	AGGAAA	2470.61	3737	1.513	0.414
RK	AGAAAA	2516.58	3482	1.384	0.325
RK	CGCAAG	2954.85	2981	1.009	0.009
RK	CGGAAG	3231.73	3225	0.998	−0.002
RK	AGAAAG	3259.25	2909	0.893	−0.114
RK	CGAAAA	1352.67	1189	0.879	−0.129
RK	CGGAAA	2495.33	1834	0.735	−0.308
RK	CGAAAG	1751.85	1265	0.722	−0.326
RK	CGTAAA	976.81	566	0.579	−0.546
RK	CGCAAA	2281.54	1209	0.530	−0.635
RK	CGTAAG	1265.08	503	0.398	−0.922
RL	CGCCTC	1491.12	2511	1.684	0.521
RL	CGCCTG	3098.73	4809	1.552	0.439
RL	CGGCTG	3389.08	5029	1.484	0.395
RL	CGGCTC	1630.84	2301	1.411	0.344
RL	CGTTTA	256.76	337	1.313	0.272
RL	AGATTA	661.49	862	1.303	0.265
RL	CGTCTT	440.20	562	1.277	0.244
RL	CGTCTA	237.40	296	1.247	0.221
RL	CGTTTG	431.33	526	1.219	0.198
RL	CGTCTC	638.40	723	1.133	0.124
RL	AGGCTA	600.44	669	1.114	0.108
RL	AGACTT	1134.11	1227	1.082	0.079
RL	AGGCTG	3355.51	3531	1.052	0.051
RL	AGACTA	611.62	617	1.009	0.009
RL	AGGCTT	1113.39	1104	0.992	−0.008
RL	CGACTA	328.75	324	0.986	−0.015
RL	CGGCTA	606.45	593	0.978	−0.022
RL	CGTCTG	1326.68	1281	0.966	−0.035
RL	AGGCTC	1614.68	1540	0.954	−0.047
RL	CGATTA	355.55	337	0.948	−0.054
RL	CGACTT	609.59	576	0.945	−0.057
RL	CGCCTA	554.49	501	0.904	−0.101
RL	AGGTTA	649.40	586	0.902	−0.103
RL	CGCCTT	1028.19	862	0.838	−0.176
RL	CGCTTG	1007.46	804	0.798	−0.226
RL	CGGCTT	1124.53	866	0.770	−0.261
RL	AGATTG	1111.24	839	0.755	−0.281
RL	CGACTC	884.04	663	0.750	−0.288
RL	AGGTTG	1090.94	774	0.709	−0.343
RL	AGACTC	1644.73	1142	0.694	−0.365
RL	CGATTG	597.29	408	0.683	−0.381
RL	CGACTG	1837.15	1128	0.614	−0.488
RL	CGCTTA	599.71	345	0.575	−0.553
RL	CGGTTG	1101.86	566	0.514	−0.666
RL	AGACTG	3417.95	1701	0.498	−0.698
RL	CGGTTA	655.90	297	0.453	−0.792
RM	CGCATG	1558.32	1961	1.258	0.230
RM	AGGATG	1687.45	1974	1.170	0.157
RM	CGAATG	923.88	932	1.009	0.009
RM	AGAATG	1718.85	1690	0.983	−0.017
RM	CGGATG	1704.33	1374	0.806	−0.215
RM	CGTATG	667.17	329	0.493	−0.707
RN	AGAAAT	1568.88	2627	1.674	0.515
RN	AGGAAC	1696.37	2200	1.297	0.260
RN	AGGAAT	1540.22	1796	1.166	0.154
RN	AGAAAC	1727.93	1949	1.128	0.120
RN	CGAAAT	843.28	930	1.103	0.098
RN	CGCAAC	1566.55	1575	1.005	0.005
RN	CGGAAC	1713.34	1621	0.946	−0.055
RN	CGAAAC	928.77	784	0.844	−0.169
RN	CGGAAT	1555.63	1002	0.644	−0.440
RN	CGTAAT	608.96	340	0.558	−0.583
RN	CGCAAT	1422.36	711	0.500	−0.693
RN	CGTAAC	670.70	308	0.459	−0.778
RP	CGGCCG	587.88	1226	2.085	0.735
RP	CGGCCC	1622.47	2939	1.811	0.594
RP	CGCCCG	537.51	717	1.334	0.288
RP	AGGCCC	1606.39	1982	1.234	0.210
RP	AGGCCG	582.05	666	1.144	0.135
RP	AGGCCT	1438.75	1642	1.141	0.132
RP	AGGCCA	1388.57	1511	1.088	0.084
RP	CGTCCT	568.84	589	1.035	0.035
RP	AGACCA	1414.41	1387	0.981	−0.020
RP	CGGCCT	1453.14	1390	0.957	−0.044
RP	AGACCT	1465.52	1398	0.954	−0.047
RP	CGTCCC	635.12	582	0.916	−0.087
RP	CGGCCA	1402.47	1285	0.916	−0.087
RP	CGCCCC	1483.46	1320	0.890	−0.117
RP	CGTCCA	549.00	487	0.887	−0.120
RP	AGACCC	1636.29	1283	0.784	−0.243
RP	CGACCA	760.25	591	0.777	−0.252
RP	CGACCC	879.51	671	0.763	−0.271
RP	CGACCT	787.72	580	0.736	−0.306
RP	CGCCCA	1282.31	887	0.692	−0.369
RP	CGTCCG	230.13	159	0.691	−0.370
RP	CGCCCT	1328.65	830	0.625	−0.470
RP	CGACCG	318.68	184	0.577	−0.549
RP	AGACCG	592.88	246	0.415	−0.880
RQ	AGACAA	1054.78	1456	1.380	0.322
RQ	CGGCAG	2920.52	3950	1.352	0.302
RQ	CGCCAG	2670.31	3160	1.183	0.168
RQ	AGGCAA	1035.51	1177	1.137	0.128
RQ	AGGCAG	2891.59	3013	1.042	0.041
RQ	CGACAA	566.95	522	0.921	−0.083
RQ	CGTCAG	1143.25	953	0.834	−0.182
RQ	CGTCAA	409.41	327	0.799	−0.225
RQ	CGACAG	1583.16	1249	0.789	−0.237
RQ	CGGCAA	1045.87	763	0.730	−0.315
RQ	AGACAG	2945.39	2062	0.700	−0.357
RQ	CGCCAA	956.27	591	0.618	−0.481
RR	CGCCGC	1172.08	2232	1.904	0.644
RR	CGGCGG	1402.02	2316	1.652	0.502
RR	AGAAGA	1426.00	2307	1.618	0.481
RR	CGGCGC	1281.90	2064	1.610	0.476
RR	AGGAGG	1374.38	1973	1.436	0.362
RR	CGCCGG	1281.90	1679	1.310	0.270
RR	CGAAGA	766.48	987	1.288	0.253
RR	AGGAGA	1399.95	1758	1.256	0.228
RR	CGCAGG	1269.20	1565	1.233	0.209
RR	CGGAGG	1388.13	1670	1.203	0.185
RR	CGTCGT	214.84	228	1.061	0.059
RR	CGAAGG	752.48	770	1.023	0.023
RR	CGCCGT	501.81	502	1.000	0.000
RR	AGAAGG	1399.95	1325	0.946	−0.055
RR	CGGCGT	548.83	498	0.907	−0.097
RR	CGTCGA	297.51	265	0.891	−0.116
RR	CGGCGA	760.01	675	0.888	−0.119
RR	CGTCGC	501.81	438	0.873	−0.136
RR	AGGCGG	1388.13	1177	0.848	−0.165
RR	CGTCGG	548.83	450	0.820	−0.199
RR	CGACGT	297.51	241	0.810	−0.211
RR	CGCCGA	694.89	547	0.787	−0.239
RR	AGGCGA	752.48	570	0.757	−0.278
RR	CGGAGA	1413.96	1068	0.755	−0.281
RR	AGACGA	766.48	557	0.727	−0.319
RR	AGGCGT	543.39	383	0.705	−0.350
RR	AGGCGC	1269.20	889	0.700	−0.356
RR	AGACGT	553.50	376	0.679	−0.387
RR	CGACGA	411.98	272	0.660	−0.415
RR	CGCAGA	1292.82	771	0.596	−0.517
RR	CGACGG	760.01	411	0.541	−0.615
RR	CGACGC	694.89	368	0.530	−0.636
RR	CGTAGA	553.50	271	0.490	−0.714
RR	CGTAGG	543.39	235	0.432	−0.838
RR	AGACGC	1292.82	524	0.405	−0.903
RR	AGACGG	1413.96	569	0.402	−0.910
RS	CGCTCG	332.61	817	2.456	0.899
RS	CGCAGC	1425.00	2853	2.002	0.694
RS	CGCTCC	1257.78	2184	1.736	0.552
RS	AGAAGT	991.66	1532	1.545	0.435
RS	CGTTCT	468.44	687	1.467	0.383
RS	CGAAGT	533.02	728	1.366	0.312
RS	CGTTCC	538.50	707	1.313	0.272
RS	AGGAGC	1543.09	1992	1.291	0.255
RS	CGTTCA	378.71	471	1.244	0.218
RS	CGGAGC	1558.53	1856	1.191	0.175
RS	AGGAGT	973.54	1071	1.100	0.095
RS	AGAAGC	1571.80	1628	1.036	0.035
RS	AGATCA	975.67	1000	1.025	0.025
RS	CGAAGC	844.85	859	1.017	0.017
RS	CGCTCA	884.55	860	0.972	−0.028
RS	CGCAGT	899.04	853	0.949	−0.053
RS	AGATCT	1206.86	1106	0.916	−0.087
RS	CGCTCT	1094.14	942	0.861	−0.150
RS	CGTTCG	142.40	121	0.850	−0.163
RS	AGGTCA	957.85	808	0.844	−0.170
RS	CGATCA	524.43	416	0.793	−0.232
RS	AGGTCT	1184.81	939	0.793	−0.233
RS	AGGTCG	360.17	284	0.789	−0.238
RS	CGATCT	648.69	497	0.766	−0.266
RS	AGGTCC	1362.00	1036	0.761	−0.274
RS	CGGAGT	983.28	745	0.758	−0.278
RS	CGTAGT	384.91	278	0.722	−0.325
RS	CGGTCG	363.77	235	0.646	−0.437
RS	CGATCC	745.70	455	0.610	−0.494
RS	AGATCC	1387.35	830	0.598	−0.514
RS	CGGTCC	1375.63	821	0.597	−0.516
RS	CGATCG	197.19	107	0.543	−0.611
RS	CGGTCA	967.43	507	0.524	−0.646
RS	CGTAGC	610.09	317	0.520	−0.655
RS	AGATCG	366.87	177	0.482	−0.729
RS	CGGTCT	1196.66	518	0.433	−0.837
RT	CGCACG	450.78	858	1.903	0.644
RT	AGAACT	1083.61	1467	1.354	0.303
RT	CGCACC	1372.27	1821	1.327	0.283
RT	AGGACG	488.14	646	1.323	0.280
RT	AGGACT	1063.81	1389	1.306	0.267
RT	AGAACA	1225.34	1575	1.285	0.251
RT	AGGACA	1202.96	1523	1.266	0.236
RT	AGGACC	1485.98	1773	1.193	0.177
RT	CGGACG	493.02	537	1.089	0.085
RT	CGAACA	658.62	661	1.004	0.004
RT	CGAACT	582.44	556	0.955	−0.046
RT	CGGACC	1500.85	1408	0.938	−0.064
RT	CGCACA	1110.90	984	0.886	−0.121
RT	CGGACA	1215.00	949	0.781	−0.247
RT	AGAACC	1513.63	1166	0.770	−0.261
RT	CGTACT	420.60	313	0.744	−0.295
RT	CGAACC	813.58	599	0.736	−0.306
RT	CGGACT	1074.45	712	0.663	−0.411
RT	CGCACT	982.40	638	0.649	−0.432
RT	CGTACC	587.52	361	0.614	−0.487
RT	AGAACG	497.22	302	0.607	−0.499
RT	CGTACA	475.62	288	0.606	−0.502
RT	CGAACG	267.26	154	0.576	−0.551
RT	CGTACG	193.00	79	0.409	−0.893
RV	CGTGTG	889.90	1699	1.909	0.647
RV	CGTGTC	449.83	826	1.836	0.608
RV	CGAGTA	315.92	562	1.779	0.576
RV	CGTGTA	228.14	391	1.714	0.539
RV	CGTGTT	351.34	565	1.608	0.475
RV	AGAGTT	905.17	1350	1.491	0.400
RV	AGAGTA	587.76	876	1.490	0.399
RV	CGAGTC	622.91	914	1.467	0.383
RV	CGAGTT	486.53	681	1.400	0.336
RV	CGAGTG	1232.31	1576	1.279	0.246
RV	CGGGTC	1149.12	1310	1.140	0.131
RV	AGGGTC	1137.73	1221	1.073	0.071
RV	CGGGTG	2273.30	2328	1.024	0.024
RV	AGAGTC	1158.91	1154	0.996	−0.004
RV	CGCGTG	2078.54	1725	0.830	−0.186
RV	AGGGTA	577.02	471	0.816	−0.203
RV	AGAGTG	2292.67	1750	0.763	−0.270
RV	CGGGTA	582.79	438	0.752	−0.286
RV	AGGGTG	2250.78	1658	0.737	−0.306
RV	CGCGTC	1050.67	763	0.726	−0.320
RV	AGGGTT	888.63	645	0.726	−0.320
RV	CGGGTT	897.52	548	0.611	−0.493
RV	CGCGTA	532.86	132	0.248	−1.395
RV	CGCGTT	820.63	178	0.217	−1.528
RW	CGCTGG	1038.00	2199	2.118	0.751
RW	CGTTGG	444.40	380	0.855	−0.157
RW	AGGTGG	1124.01	876	0.779	−0.249
RW	CGATGG	615.40	466	0.757	−0.278
RW	AGATGG	1144.93	804	0.702	−0.353
RW	CGGTGG	1135.26	777	0.684	−0.379
RY	CGCTAC	1173.12	2612	2.227	0.800
RY	CGCTAT	953.25	1198	1.257	0.229
RY	CGTTAC	502.25	565	1.125	0.118
RY	CGTTAT	408.12	459	1.125	0.117
RY	AGATAT	1051.45	1018	0.968	−0.032
RY	AGATAC	1293.97	1239	0.958	−0.043
RY	CGATAT	565.15	509	0.901	−0.105
RY	CGATAC	695.51	584	0.840	−0.175
RY	AGGTAC	1270.33	1007	0.793	−0.232
RY	AGGTAT	1032.24	769	0.745	−0.294
RY	CGGTAC	1283.04	856	0.667	−0.405
RY	CGGTAT	1042.57	455	0.436	−0.829
SA	TCGGCG	241.39	778	3.223	1.170
SA	TCGGCC	892.76	1976	2.213	0.795
SA	TCAGCA	1366.87	2526	1.848	0.614
SA	TCTGCA	1690.75	3035	1.795	0.585
SA	TCTGCT	1931.41	3350	1.734	0.551
SA	TCAGCT	1561.43	2630	1.684	0.521
SA	AGTGCT	1587.01	2487	1.567	0.449
SA	AGTGCA	1389.27	2040	1.468	0.384
SA	AGTGCC	2413.15	3437	1.424	0.354
SA	TCAGCC	2374.25	3294	1.387	0.327
SA	TCGGCT	587.12	808	1.376	0.319
SA	TCTGCC	2936.83	3480	1.185	0.170
SA	TCGGCA	513.97	598	1.163	0.151
SA	TCTGCG	794.06	745	0.938	−0.064
SA	TCAGCG	641.95	584	0.910	−0.095
SA	AGTGCG	652.47	532	0.815	−0.204
SA	AGCGCG	1034.18	802	0.775	−0.254
SA	AGCGCC	3824.90	2428	0.635	−0.454
SA	TCCGCG	912.82	577	0.632	−0.459
SA	TCCGCC	3376.05	1230	0.364	−1.010
SA	AGCGCT	2515.45	709	0.282	−1.266
SA	AGCGCA	2202.02	601	0.273	−1.299
SA	TCCGCA	1943.61	476	0.245	−1.407
SA	TCCGCT	2220.26	481	0.217	−1.530
SC	TCCTGC	1640.34	2828	1.724	0.545
SC	AGCTGC	1858.43	3034	1.633	0.490
SC	TCCTGT	1381.63	1779	1.288	0.253
SC	AGCTGT	1565.33	1922	1.228	0.205
SC	TCGTGC	433.77	361	0.832	−0.184
SC	TCTTGT	1201.89	941	0.783	−0.245
SC	AGTTGT	987.57	698	0.707	−0.347
SC	TCGTGT	365.36	225	0.616	−0.485
SC	TCATGT	971.65	584	0.601	−0.509
SC	TCTTGC	1426.94	758	0.531	−0.633
SC	TCATGC	1153.59	525	0.455	−0.787
SC	AGTTGC	1172.49	504	0.430	−0.844
SD	TCAGAT	1978.63	3706	1.873	0.628
SD	AGTGAT	2011.05	3683	1.831	0.605
SD	AGTGAC	2271.71	4040	1.778	0.576
SD	TCGGAC	840.43	1438	1.711	0.537
SD	TCTGAT	2447.46	3578	1.462	0.380
SD	TCAGAC	2235.09	2906	1.300	0.262
SD	TCGGAT	744.00	840	1.129	0.121
SD	TCTGAC	2764.69	2949	1.067	0.065
SD	AGCGAC	3600.71	2017	0.560	−0.580
SD	TCCGAC	3178.17	1336	0.420	−0.867
SD	AGCGAT	3187.56	920	0.289	−1.243
SD	TCCGAT	2813.50	660	0.235	−1.450
SE	TCAGAA	2420.84	4815	1.989	0.688
SE	AGTGAA	2460.50	4686	1.904	0.644
SE	TCGGAG	1217.33	2184	1.794	0.584
SE	TCTGAA	2994.45	4621	1.543	0.434
SE	TCAGAG	3237.43	4683	1.447	0.369
SE	AGTGAG	3290.47	4410	1.340	0.293
SE	TCTGAG	4004.54	4891	1.221	0.200
SE	TCGGAA	910.28	879	0.966	−0.035
SE	AGCGAG	5215.47	2961	0.568	−0.566
SE	TCCGAG	4603.44	2005	0.436	−0.831
SE	AGCGAA	3899.95	847	0.217	−1.527
SE	TCCGAA	3442.29	715	0.208	−1.572
SF	TCCTTC	2645.79	4407	1.666	0.510
SF	AGCTTC	2997.56	3942	1.315	0.274
SF	TCATTT	1625.65	1773	1.091	0.087
SF	TCCTTT	2311.58	2487	1.076	0.073
SF	AGTTTT	1652.29	1695	1.026	0.026
SF	AGCTTT	2618.91	2370	0.905	−0.100
SF	TCTTTT	2010.85	1809	0.900	−0.106
SF	TCTTTC	2301.58	1728	0.751	−0.287
SF	AGTTTC	1891.18	1353	0.715	−0.335
SF	TCGTTT	611.27	342	0.559	−0.581
SF	TCATTC	1860.69	991	0.533	−0.630
SF	TCGTTC	699.65	330	0.472	−0.751
SG	AGTGGT	1051.00	2094	1.992	0.689
SG	TCGGGG	586.31	1117	1.905	0.645
SG	TCGGGC	814.29	1487	1.826	0.602
SG	AGTGGA	1623.36	2932	1.806	0.591
SG	TCAGGA	1597.19	2760	1.728	0.547
SG	TCTGGA	1975.64	3391	1.716	0.540
SG	AGTGGG	1584.81	2584	1.630	0.489
SG	TCTGGG	1928.73	2974	1.542	0.433
SG	AGTGGC	2201.05	3314	1.506	0.409
SG	TCTGGT	1279.07	1902	1.487	0.397
SG	TCAGGG	1559.26	2161	1.386	0.326
SG	TCAGGT	1034.06	1351	1.307	0.267
SG	TCGGGA	600.57	684	1.139	0.130
SG	TCGGGT	388.82	410	1.054	0.053
SG	TCTGGC	2678.70	2734	1.021	0.020
SG	TCAGGC	2165.57	2114	0.976	−0.024
SG	AGCGGC	3488.72	2475	0.709	−0.343
SG	AGCGGG	2511.96	1464	0.583	−0.540
SG	TCCGGG	2217.18	1117	0.504	−0.686
SG	TCCGGC	3079.31	1163	0.378	−0.974
SG	AGCGGT	1665.85	536	0.322	−1.134
SG	AGCGGA	2573.06	663	0.258	−1.356
SG	TCCGGA	2271.11	560	0.247	−1.400
SG	TCCGGT	1470.37	359	0.244	−1.410
SH	AGCCAC	2202.27	3210	1.458	0.377
SH	TCTCAT	1226.22	1426	1.163	0.151
SH	TCCCAC	1943.83	2233	1.149	0.139
SH	AGTCAT	1007.57	1082	1.074	0.071
SH	AGCCAT	1597.01	1606	1.006	0.006
SH	TCGCAC	514.03	512	0.996	−0.004
SH	TCCCAT	1409.60	1349	0.957	−0.044
SH	TCACAT	991.32	929	0.937	−0.065
SH	AGTCAC	1389.42	1077	0.775	−0.255
SH	TCACAC	1367.03	956	0.699	−0.358
SH	TCTCAC	1690.94	1158	0.685	−0.379
SH	TCGCAT	372.75	174	0.467	−0.762
SI	TCCATC	2374.96	4526	1.906	0.645
SI	AGCATC	2690.72	4471	1.662	0.508
SI	TCCATT	1878.09	2383	1.269	0.238
SI	AGCATT	2127.79	2384	1.120	0.114
SI	TCCATA	863.76	963	1.115	0.109
SI	AGTATA	617.40	640	1.037	0.036
SI	TCAATA	607.45	618	1.017	0.017
SI	AGTATT	1342.43	1299	0.968	−0.033
SI	AGCATA	978.60	943	0.964	−0.037
SI	TCTATA	751.38	658	0.876	−0.133
SI	TCTATT	1633.75	1215	0.744	−0.296
SI	TCAATT	1320.79	957	0.725	−0.322
SI	AGTATC	1697.59	924	0.544	−0.608
SI	TCGATA	228.41	109	0.477	−0.740
SI	TCTATC	2065.98	958	0.464	−0.769
SI	TCGATT	496.64	185	0.373	−0.988
SI	TCAATC	1670.22	557	0.333	−1.098
SI	TCGATC	628.03	184	0.293	−1.228
SK	TCCAAG	3563.99	5021	1.409	0.343
SK	TCCAAA	2751.88	3634	1.321	0.278
SK	AGCAAG	4037.83	5128	1.270	0.239
SK	AGCAAA	3117.75	3736	1.198	0.181
SK	TCAAAA	1935.30	2282	1.179	0.165
SK	AGTAAA	1967.01	2149	1.093	0.088
SK	TCAAAG	2506.42	2082	0.831	−0.186
SK	TCTAAA	2393.86	1838	0.768	−0.264
SK	TCGAAG	942.46	522	0.554	−0.591
SK	AGTAAG	2547.49	1300	0.510	−0.673
SK	TCTAAG	3100.32	1569	0.506	−0.681
SK	TCGAAA	727.71	331	0.455	−0.788
SL	AGTTTA	709.05	1103	1.556	0.442
SL	TCGCTG	1355.42	2104	1.552	0.440
SL	TCCTTG	1666.44	2462	1.477	0.390
SL	TCTTTA	862.92	1267	1.468	0.384
SL	AGCCTC	2794.39	4013	1.436	0.362
SL	TCTTTG	1449.64	2009	1.386	0.326
SL	TCATTA	697.62	862	1.236	0.212
SL	AGCCTG	5807.08	7014	1.208	0.189
SL	AGTTTG	1191.15	1427	1.198	0.181
SL	TCGCTC	652.23	777	1.191	0.175
SL	TCTCTA	797.87	950	1.191	0.175
SL	TCTCTT	1479.47	1750	1.183	0.168
SL	TCCCTG	5125.62	6034	1.177	0.163
SL	TCCCTC	2466.46	2805	1.137	0.129
SL	TCCTTA	991.98	1076	1.085	0.081
SL	AGTCTT	1215.66	1242	1.022	0.021
SL	AGCCTT	1926.85	1959	1.017	0.017
SL	TCACTA	645.03	630	0.977	−0.024
SL	AGCTTG	1888.00	1786	0.946	−0.056
SL	TCACTT	1196.06	1111	0.929	−0.074
SL	TCCCTT	1700.73	1545	0.908	−0.096
SL	TCCCTA	917.19	810	0.883	−0.124
SL	AGTCTA	655.60	569	0.868	−0.142
SL	TCATTG	1171.95	1015	0.866	−0.144
SL	AGCCTA	1039.14	875	0.842	−0.172
SL	TCTCTC	2145.58	1760	0.820	−0.198
SL	TCTCTG	4458.78	3418	0.767	−0.266
SL	AGCTTA	1123.86	758	0.674	−0.394
SL	AGTCTC	1763.00	1158	0.657	−0.420
SL	TCGTTG	440.67	280	0.635	−0.454
SL	TCACTC	1734.58	1100	0.634	−0.455
SL	TCACTG	3604.66	2254	0.625	−0.470
SL	TCGCTT	449.74	279	0.620	−0.477
SL	TCGCTA	242.54	143	0.590	−0.528
SL	TCGTTA	262.32	140	0.534	−0.628
SL	AGTCTG	3663.72	1808	0.493	−0.706
SM	TCCATG	2282.65	3908	1.712	0.538
SM	AGCATG	2586.13	3300	1.276	0.244
SM	TCAATG	1605.31	1129	0.703	−0.352
SM	TCGATG	603.62	365	0.605	−0.503
SM	AGTATG	1631.61	966	0.592	−0.524
SM	TCTATG	1985.68	1027	0.517	−0.659
SN	AGCAAC	2539.42	3717	1.464	0.381
SN	TCCAAC	2241.42	3216	1.435	0.361
SN	TCAAAT	1431.22	1883	1.316	0.274
SN	AGCAAT	2305.68	2513	1.090	0.086
SN	TCCAAT	2035.11	2000	0.983	−0.017
SN	AGTAAT	1454.67	1425	0.980	−0.021
SN	AGTAAC	1602.14	1339	0.836	−0.179
SN	TCAAAC	1576.31	1194	0.757	−0.278
SN	TCTAAT	1770.34	1297	0.733	−0.311
SN	TCTAAC	1949.81	955	0.490	−0.714
SN	TCGAAT	538.16	258	0.479	−0.735
SN	TCGAAC	592.72	240	0.405	−0.904
SP	TCGCCG	282.21	549	1.945	0.665
SP	TCGCCC	778.87	1221	1.568	0.450
SP	TCCCCG	1067.21	1621	1.519	0.418
SP	TCTCCA	2214.76	3119	1.408	0.342
SP	AGCCCC	3336.96	4654	1.395	0.333
SP	TCTCCT	2294.78	2888	1.259	0.230
SP	AGCCCG	1209.10	1432	1.184	0.169
SP	TCCCCA	2545.99	2968	1.166	0.153
SP	TCACCA	1790.50	1869	1.044	0.043
SP	AGCCCT	2988.71	3086	1.033	0.032
SP	AGTCCT	1885.59	1904	1.010	0.010
SP	TCACCT	1855.20	1752	0.944	−0.057
SP	AGCCCA	2884.48	2607	0.904	−0.101
SP	TCCCCT	2637.98	2238	0.848	−0.164
SP	AGTCCA	1819.84	1473	0.809	−0.211
SP	TCGCCT	697.59	562	0.806	−0.216
SP	TCGCCA	673.26	541	0.804	−0.219
SP	TCTCCC	2562.18	2036	0.795	−0.230
SP	TCACCC	2071.37	1568	0.757	−0.278
SP	AGTCCC	2105.31	1534	0.729	−0.317
SP	TCTCCG	928.37	664	0.715	−0.335
SP	TCCCCC	2945.37	2058	0.699	−0.358
SP	TCACCG	750.53	426	0.568	−0.566
SP	AGTCCG	762.83	319	0.418	−0.872
SQ	TCCCAG	4427.95	5592	1.263	0.233
SQ	AGCCAG	5016.65	6041	1.204	0.186
SQ	TCTCAA	1379.40	1644	1.192	0.175
SQ	AGTCAA	1133.44	1293	1.141	0.132
SQ	TCACAA	1115.16	1196	1.072	0.070
SQ	AGCCAA	1796.52	1819	1.013	0.012
SQ	TCCCAA	1585.70	1474	0.930	−0.073
SQ	TCTCAG	3851.88	3430	0.890	−0.116
SQ	TCGCAG	1170.92	1015	0.867	−0.143
SQ	TCACAG	3114.02	2271	0.729	−0.316
SQ	AGTCAG	3165.04	2215	0.700	−0.357
SQ	TCGCAA	419.32	186	0.444	−0.813
SR	AGCCGC	1540.23	2828	1.836	0.608
SR	TCCAGG	1472.14	2309	1.568	0.450
SR	AGCCGG	1684.56	2353	1.397	0.334
SR	TCCCGG	1486.87	1976	1.329	0.284
SR	AGCAGG	1667.87	2186	1.311	0.271
SR	AGCCGT	659.43	857	1.300	0.262
SR	TCGCGC	359.50	446	1.241	0.216
SR	TCCAGA	1499.54	1850	1.234	0.210
SR	TCAAGA	1054.57	1294	1.227	0.205
SR	TCGCGG	393.19	481	1.223	0.202
SR	TCCCGC	1359.49	1605	1.181	0.166
SR	TCTCGA	701.14	826	1.178	0.164
SR	AGTCGT	416.04	484	1.163	0.151
SR	TCCCGA	806.00	937	1.163	0.151
SR	AGCAGA	1698.90	1925	1.133	0.125
SR	AGCCGA	913.16	1020	1.117	0.111
SR	TCTCGT	506.32	493	0.974	−0.027
SR	AGTCGA	576.12	553	0.960	−0.041
SR	TCCCGT	582.04	553	0.950	−0.051
SR	TCAAGG	1035.31	922	0.891	−0.116
SR	TCGAGG	389.29	324	0.832	−0.184
SR	TCTCGG	1293.43	1062	0.821	−0.197
SR	TCACGT	409.33	323	0.789	−0.237
SR	AGTAGA	1071.85	746	0.696	−0.362
SR	TCGCGT	153.92	102	0.663	−0.411
SR	AGTCGG	1062.80	675	0.635	−0.454
SR	AGTCGC	971.74	591	0.608	−0.497
SR	TCACGA	566.83	344	0.607	−0.499
SR	TCGAGA	396.54	240	0.605	−0.502
SR	TCTAGA	1304.45	750	0.575	−0.553
SR	TCGCGA	213.14	115	0.540	−0.617
SR	TCTCGC	1182.62	636	0.538	−0.620
SR	TCACGG	1045.66	534	0.511	−0.672
SR	TCTAGG	1280.62	574	0.448	−0.802
SR	TCACGC	956.08	406	0.425	−0.856
SR	AGTAGG	1052.27	443	0.421	−0.865
SS	AGCAGC	3919.72	7160	1.827	0.602
SS	TCGTCG	213.54	376	1.761	0.566
SS	TCCTCG	807.53	1302	1.612	0.478
SS	TCCAGC	3459.74	4832	1.397	0.334
SS	TCTTCA	1868.19	2596	1.390	0.329
SS	AGCAGT	2472.97	3417	1.382	0.323
SS	TCCTCC	3053.74	4162	1.363	0.310
SS	TCTTCT	2310.85	2896	1.253	0.226
SS	TCCAGT	2182.77	2691	1.233	0.209
SS	TCATCA	1510.32	1795	1.188	0.173
SS	AGCTCC	3459.74	4024	1.163	0.151
SS	TCATCT	1868.19	2118	1.134	0.126
SS	TCCTCA	2147.58	2413	1.124	0.117
SS	AGCTCG	914.89	1001	1.094	0.090
SS	TCCTCT	2656.45	2744	1.033	0.032
SS	TCGTCC	807.53	818	1.013	0.013
SS	TCTTCC	2656.45	2600	0.979	−0.021
SS	AGTTCT	1898.79	1856	0.977	−0.023
SS	AGTTCA	1535.06	1498	0.976	−0.024
SS	TCAAGT	1535.06	1404	0.915	−0.089
SS	AGCTCA	2433.11	2075	0.853	−0.159
SS	AGCTCT	3009.63	2465	0.819	−0.200
SS	TCTTCG	702.47	556	0.791	−0.234
SS	TCATCC	2147.58	1632	0.760	−0.275
SS	AGTAGT	1560.21	1030	0.660	−0.415
SS	AGTTCC	2182.77	1405	0.644	−0.441
SS	TCGTCT	702.47	434	0.618	−0.482
SS	TCATCG	567.91	343	0.604	−0.504
SS	TCGTCA	567.91	313	0.551	−0.596
SS	TCTAGT	1898.79	957	0.504	−0.685
SS	TCGAGC	914.89	440	0.481	−0.732
SS	AGTAGC	2472.97	1158	0.468	−0.759
SS	TCAAGC	2433.11	1117	0.459	−0.779
SS	TCGAGT	577.21	259	0.449	−0.801
SS	AGTTCG	577.21	251	0.435	−0.833
SS	TCTAGC	3009.63	899	0.299	−1.208
ST	TCCACG	785.52	1434	1.826	0.602
ST	AGCACC	2709.18	4149	1.531	0.426
ST	TCCACC	2391.25	3527	1.475	0.389
ST	AGCACG	889.95	1180	1.326	0.282
ST	AGCACA	2193.18	2692	1.227	0.205
ST	TCCACA	1935.81	2329	1.203	0.185
ST	TCCACT	1711.89	1937	1.131	0.124
ST	AGCACT	1939.49	2193	1.131	0.123
ST	TCAACA	1361.39	1485	1.091	0.087
ST	TCAACT	1203.91	1270	1.055	0.053
ST	TCTACT	1489.18	1390	0.933	−0.069
ST	TCTACA	1683.97	1461	0.868	−0.142
ST	AGTACT	1223.64	1036	0.847	−0.166
ST	AGTACA	1383.69	1061	0.767	−0.266
ST	TCGACG	207.72	145	0.698	−0.359
ST	TCTACC	2080.15	1218	0.586	−0.535
ST	TCGACC	632.34	365	0.577	−0.550
ST	AGTACC	1709.24	976	0.571	−0.560
ST	TCGACT	452.69	240	0.530	−0.635
ST	TCAACC	1681.68	873	0.519	−0.656
ST	TCAACG	552.43	275	0.498	−0.698
ST	TCGACA	511.90	236	0.461	−0.774
ST	TCTACG	683.32	302	0.442	−0.817
ST	AGTACG	561.48	201	0.358	−1.027
SV	TCGGTG	935.47	1822	1.948	0.667
SV	TCTGTA	788.92	1398	1.772	0.572
SV	TCTGTT	1214.96	2136	1.758	0.564
SV	TCAGTA	637.79	1121	1.758	0.564
SV	AGTGTT	998.32	1719	1.722	0.543
SV	TCAGTT	982.23	1591	1.620	0.482
SV	TCTGTC	1555.54	2367	1.522	0.420
SV	AGTGTC	1278.17	1943	1.520	0.419
SV	TCTGTG	3077.33	4672	1.518	0.418
SV	AGTGTA	648.24	976	1.506	0.409
SV	TCGGTC	472.87	683	1.444	0.368
SV	TCAGTG	2487.84	2925	1.176	0.162
SV	AGTGTG	2528.60	2901	1.147	0.137
SV	TCAGTC	1257.56	1351	1.074	0.072
SV	TCGGTA	239.82	231	0.963	−0.037
SV	TCGGTT	369.33	266	0.720	−0.328
SV	AGCGTC	2025.93	1298	0.641	−0.445
SV	TCCGTG	3537.57	2065	0.584	−0.538
SV	AGCGTG	4007.89	2221	0.554	−0.590
SV	TCCGTC	1788.18	829	0.464	−0.769
SV	AGCGTT	1582.36	446	0.282	−1.266
SV	TCCGTA	906.91	239	0.264	−1.334
SV	TCCGTT	1396.67	329	0.236	−1.446
SV	AGCGTA	1027.48	217	0.211	−1.555
SW	TCCTGG	1756.97	2825	1.608	0.475
SW	AGCTGG	1990.56	2404	1.208	0.189
SW	TCGTGG	464.61	444	0.956	−0.045
SW	TCTTGG	1528.39	1137	0.744	−0.296
SW	TCATGG	1235.61	778	0.630	−0.463
SW	AGTTGG	1255.86	644	0.513	−0.668
SY	TCCTAC	1871.53	3038	1.623	0.484
SY	AGCTAC	2120.35	2864	1.351	0.301
SY	TCCTAT	1520.75	1869	1.229	0.206
SY	AGCTAT	1722.94	1609	0.934	−0.068
SY	AGTTAT	1087.01	1010	0.929	−0.073
SY	AGTTAC	1337.74	1153	0.862	−0.149
SY	TCATAT	1069.49	897	0.839	−0.176
SY	TCTTAT	1322.91	1100	0.832	−0.185
SY	TCTTAC	1628.04	1204	0.740	−0.302
SY	TCGTAC	494.91	304	0.614	−0.487
SY	TCGTAT	402.15	204	0.507	−0.679
SY	TCATAC	1316.18	642	0.488	−0.718
TA	ACGGCG	348.71	734	2.105	0.744
TA	ACAGCA	1829.79	3283	1.794	0.585
TA	ACGGCC	1289.71	2090	1.621	0.483
TA	ACTGCA	1618.13	2557	1.580	0.458
TA	ACAGCT	2090.24	3295	1.576	0.455
TA	ACTGCT	1848.45	2764	1.495	0.402
TA	ACAGCC	3178.34	3912	1.231	0.208
TA	ACGGCA	742.49	804	1.083	0.080
TA	ACTGCC	2810.69	3015	1.073	0.070
TA	ACGGCT	848.18	804	0.948	−0.053
TA	ACAGCG	859.36	803	0.934	−0.068
TA	ACTGCG	759.96	623	0.820	−0.199
TA	ACCGCG	1061.55	584	0.550	−0.598
TA	ACCGCC	3926.11	1648	0.420	−0.868
TA	ACCGCA	2260.29	561	0.248	−1.394
TA	ACCGCT	2582.01	577	0.223	−1.498
TC	ACCTGC	1892.82	3247	1.715	0.540
TC	ACCTGT	1594.30	1994	1.251	0.224
TC	ACGTGC	621.78	691	1.111	0.106
TC	ACGTGT	523.72	484	0.924	−0.079
TC	ACTTGT	1141.35	1033	0.905	−0.100
TC	ACATGT	1290.64	938	0.727	−0.319
TC	ACTTGC	1355.07	815	0.601	−0.508
TC	ACATGC	1532.31	750	0.489	−0.714
TD	ACAGAT	2415.25	4195	1.737	0.552
TD	ACAGAC	2728.31	3765	1.380	0.322
TD	ACTGAT	2135.87	2913	1.364	0.310
TD	ACGGAC	1107.10	1446	1.306	0.267
TD	ACTGAC	2412.71	2615	1.084	0.081
TD	ACGGAT	980.07	922	0.941	−0.061
TD	ACCGAC	3370.20	1547	0.459	−0.779
TD	ACCGAT	2983.49	730	0.245	−1.408
TE	ACAGAA	3127.33	5307	1.697	0.529
TE	ACGGAG	1697.07	2517	1.483	0.394
TE	ACTGAA	2765.58	4093	1.480	0.392
TE	ACAGAG	4182.23	5419	1.296	0.259
TE	ACTGAG	3698.46	4124	1.115	0.109
TE	ACGGAA	1269.01	1080	0.851	−0.161
TE	ACCGAG	5166.20	2450	0.474	−0.746
TE	ACCGAA	3863.10	779	0.202	−1.601
TF	ACCTTC	3026.54	4955	1.637	0.493
TF	ACATTT	2140.61	2275	1.063	0.061
TF	ACTTTT	1893.00	1904	1.006	0.006
TF	ACCTTT	2644.23	2518	0.952	−0.049
TF	ACTTTC	2166.69	1822	0.841	−0.173
TF	ACGTTT	868.62	650	0.748	−0.290
TF	ACGTTC	994.21	666	0.670	−0.401
TF	ACATTC	2450.10	1394	0.569	−0.564
TG	ACTGGA	1710.74	3660	2.139	0.761
TG	ACTGGT	1107.57	1887	1.704	0.533
TG	ACAGGA	1934.51	2970	1.535	0.429
TG	ACGGGC	1064.34	1583	1.487	0.397
TG	ACTGGG	1670.12	2322	1.390	0.330
TG	ACGGGG	766.35	1049	1.369	0.314
TG	ACAGGT	1252.44	1694	1.353	0.302
TG	ACAGGG	1888.57	2148	1.137	0.129
TG	ACTGGC	2319.53	2620	1.130	0.122
TG	ACAGGC	2622.93	2664	1.016	0.016
TG	ACGGGT	508.22	484	0.952	−0.049
TG	ACGGGA	784.99	710	0.904	−0.100
TG	ACCGGG	2332.90	1093	0.469	−0.758
TG	ACCGGC	3240.03	1373	0.424	−0.859
TG	ACCGGT	1547.11	355	0.229	−1.472
TG	ACCGGA	2389.65	528	0.221	−1.510
TH	ACTCAT	1054.95	1291	1.224	0.202
TH	ACCCAC	2032.09	2408	1.185	0.170
TH	ACGCAC	667.53	764	1.145	0.135
TH	ACACAT	1192.94	1186	0.994	−0.006
TH	ACTCAC	1454.76	1384	0.951	−0.050
TH	ACCCAT	1473.60	1287	0.873	−0.135
TH	ACACAC	1645.05	1383	0.841	−0.174
TH	ACGCAT	484.07	302	0.624	−0.472
TI	ACCATC	2842.70	5915	2.081	0.733
TI	ACCATT	2247.97	2878	1.280	0.247
TI	ACAATA	836.96	980	1.171	0.158
TI	ACCATA	1033.87	1137	1.100	0.095
TI	ACAATT	1819.82	1579	0.868	−0.142
TI	ACTATA	740.14	642	0.867	−0.142
TI	ACTATT	1609.31	1337	0.831	−0.185
TI	ACGATA	339.62	190	0.559	−0.581
TI	ACGATT	738.45	389	0.527	−0.641
TI	ACGATC	933.81	463	0.496	−0.702
TI	ACTATC	2035.08	942	0.463	−0.770
TI	ACAATC	2301.27	1027	0.446	−0.807
TK	ACCAAG	3878.56	6678	1.722	0.543
TK	ACCAAA	2994.77	3789	1.265	0.235
TK	ACAAAA	2424.38	2546	1.050	0.049
TK	ACAAAG	3139.84	2507	0.798	−0.225
TK	ACTAAA	2143.95	1684	0.785	−0.241
TK	ACGAAG	1274.09	708	0.556	−0.588
TK	ACGAAA	983.77	511	0.519	−0.655
TK	ACTAAG	2776.65	1193	0.430	−0.845
TL	ACGCTG	1815.48	3357	1.849	0.615
TL	ACTTTA	765.72	1207	1.576	0.455
TL	ACTTTG	1286.34	1876	1.458	0.377
TL	ACATTA	865.87	1115	1.288	0.253
TL	ACCTTG	1796.82	2257	1.256	0.228
TL	ACTCTA	707.99	876	1.237	0.213
TL	ACGCTC	873.61	1057	1.210	0.191
TL	ACCCTC	2659.44	3133	1.178	0.164
TL	ACCCTG	5526.65	6354	1.150	0.140
TL	ACTCTT	1312.81	1469	1.119	0.112
TL	ACACTA	800.60	799	0.998	−0.002
TL	ACGCTA	324.87	307	0.945	−0.057
TL	ACCTTA	1069.59	957	0.895	−0.111
TL	ACACTT	1484.53	1316	0.886	−0.121
TL	ACGTTG	590.25	505	0.856	−0.156
TL	ACATTG	1454.60	1210	0.832	−0.184
TL	ACCCTT	1833.80	1515	0.826	−0.191
TL	ACCCTA	988.95	802	0.811	−0.210
TL	ACTCTG	3956.51	3120	0.789	−0.238
TL	ACGTTA	351.36	262	0.746	−0.293
TL	ACTCTC	1903.88	1391	0.731	−0.314
TL	ACGCTT	602.39	427	0.709	−0.344
TL	ACACTG	4474.03	3013	0.673	−0.395
TL	ACACTC	2152.92	1274	0.592	−0.525
TM	ACCATG	2733.42	4467	1.634	0.491
TM	ACAATG	2212.81	1641	0.742	−0.299
TM	ACGATG	897.92	655	0.729	−0.315
TM	ACTATG	1956.85	1038	0.530	−0.634
TN	ACCAAC	2378.62	4300	1.808	0.592
TN	ACAAAT	1748.34	2194	1.255	0.227
TN	ACCAAT	2159.68	2454	1.136	0.128
TN	ACAAAC	1925.59	1486	0.772	−0.259
TN	ACTAAT	1546.11	1077	0.697	−0.362
TN	ACGAAT	709.45	336	0.474	−0.747
TN	ACTAAC	1702.85	789	0.463	−0.769
TN	ACGAAC	781.37	316	0.404	−0.905
TP	ACGCCG	349.03	632	1.811	0.594
TP	ACGCCC	963.29	1491	1.548	0.437
TP	ACTCCA	1814.66	2359	1.300	0.262
TP	ACCCCG	1062.52	1331	1.253	0.225
TP	ACTCCT	1880.23	2186	1.163	0.151
TP	ACACCA	2052.02	2361	1.151	0.140
TP	ACCCCA	2534.80	2784	1.098	0.094
TP	ACACCT	2126.17	2104	0.990	−0.010
TP	ACCCCT	2626.39	2415	0.920	−0.084
TP	ACGCCA	832.67	748	0.898	−0.107
TP	ACCCCC	2932.43	2380	0.812	−0.209
TP	ACACCC	2373.91	1922	0.810	−0.211
TP	ACGCCT	862.76	697	0.808	−0.213
TP	ACTCCC	2099.31	1649	0.785	−0.241
TP	ACTCCG	760.66	538	0.707	−0.346
TP	ACACCG	860.15	534	0.621	−0.477
TQ	ACTCAA	1103.35	1368	1.240	0.215
TQ	ACCCAG	4303.71	5173	1.202	0.184
TQ	ACGCAG	1413.75	1518	1.074	0.071
TQ	ACACAA	1247.67	1328	1.064	0.062
TQ	ACTCAG	3081.01	2839	0.921	−0.082
TQ	ACCCAA	1541.21	1410	0.915	−0.089
TQ	ACACAG	3484.02	2765	0.794	−0.231
TQ	ACGCAA	506.28	280	0.553	−0.592
TR	ACCAGG	1331.08	2049	1.539	0.431
TR	ACGCGC	403.79	605	1.498	0.404
TR	ACGCGG	441.63	661	1.497	0.403
TR	ACTCGA	521.72	717	1.374	0.318
TR	ACAAGA	1097.61	1429	1.302	0.264
TR	ACCCGC	1229.22	1547	1.259	0.230
TR	ACCCGG	1344.40	1668	1.241	0.216
TR	ACTCGT	376.76	448	1.189	0.173
TR	ACCAGA	1355.85	1599	1.179	0.165
TR	ACCCGA	728.77	758	1.040	0.039
TR	ACCCGT	526.27	535	1.017	0.016
TR	ACAAGG	1077.56	1072	0.995	−0.005
TR	ACGAGG	437.25	433	0.990	−0.010
TR	ACTCGG	962.45	823	0.855	−0.157
TR	ACGCGT	172.88	141	0.816	−0.204
TR	ACACGT	426.04	329	0.772	−0.258
TR	ACGAGA	445.39	331	0.743	−0.297
TR	ACACGA	589.97	432	0.732	−0.312
TR	ACACGG	1088.34	756	0.695	−0.364
TR	ACTCGC	879.99	607	0.690	−0.371
TR	ACTAGA	970.65	624	0.643	−0.442
TR	ACGCGA	239.40	150	0.627	−0.468
TR	ACACGC	995.10	498	0.500	−0.692
TR	ACTAGG	952.91	383	0.402	−0.911
TS	ACCAGC	2807.29	4575	1.630	0.488
TS	ACCTCG	655.24	1060	1.618	0.481
TS	ACGTCG	215.24	348	1.617	0.480
TS	ACTTCA	1247.51	1844	1.478	0.391
TS	ACTTCT	1543.11	1974	1.279	0.246
TS	ACATCA	1410.69	1754	1.243	0.218
TS	ACCAGT	1771.14	2194	1.239	0.214
TS	ACCTCC	2477.85	3050	1.231	0.208
TS	ACCTCA	1742.59	1938	1.112	0.106
TS	ACATCT	1744.95	1911	1.095	0.091
TS	ACGTCC	813.96	840	1.032	0.031
TS	ACCTCT	2155.49	2072	0.961	−0.040
TS	ACAAGT	1433.80	1335	0.931	−0.071
TS	ACTTCC	1773.89	1524	0.859	−0.152
TS	ACGTCA	572.43	450	0.786	−0.241
TS	ACATCC	2005.92	1570	0.783	−0.245
TS	ACTTCG	469.09	353	0.753	−0.284
TS	ACGTCT	708.07	527	0.744	−0.295
TS	ACATCG	530.44	361	0.681	−0.385
TS	ACTAGT	1267.95	725	0.572	−0.559
TS	ACAAGC	2272.61	1275	0.561	−0.578
TS	ACGAGT	581.81	297	0.510	−0.672
TS	ACGAGC	922.18	469	0.509	−0.676
TS	ACTAGC	2009.73	687	0.342	−1.073
TT	ACCACG	875.88	1567	1.789	0.582
TT	ACCACC	2666.32	4767	1.788	0.581
TT	ACCACA	2158.49	2882	1.335	0.289
TT	ACCACT	1908.81	2309	1.210	0.190
TT	ACAACA	1747.38	1793	1.026	0.026
TT	ACAACT	1545.26	1567	1.014	0.014
TT	ACGACG	287.72	252	0.876	−0.133
TT	ACTACT	1366.51	1065	0.779	−0.249
TT	ACTACA	1545.26	1196	0.774	−0.256
TT	ACGACC	875.88	575	0.656	−0.421
TT	ACGACA	709.06	437	0.616	−0.484
TT	ACAACC	2158.49	1310	0.607	−0.499
TT	ACGACT	627.04	357	0.569	−0.563
TT	ACTACC	1908.81	992	0.520	−0.655
TT	ACAACG	709.06	365	0.515	−0.664
TT	ACTACG	627.04	283	0.451	−0.796
TV	ACTGTA	845.20	1425	1.686	0.522
TV	ACTGTT	1301.64	2058	1.581	0.458
TV	ACGGTG	1512.80	2306	1.524	0.422
TV	ACAGTA	955.76	1371	1.434	0.361
TV	ACTGTC	1666.51	2289	1.374	0.317
TV	ACAGTT	1471.90	2019	1.372	0.316
TV	ACTGTG	3296.87	4505	1.366	0.312
TV	ACGGTC	764.70	911	1.191	0.175
TV	ACAGTG	3728.11	4108	1.102	0.097
TV	ACAGTC	1884.50	1933	1.026	0.025
TV	ACGGTA	387.83	286	0.737	−0.305
TV	ACGGTT	597.27	415	0.695	−0.364
TV	ACCGTG	4605.23	2640	0.573	−0.556
TV	ACCGTC	2327.87	1285	0.552	−0.594
TV	ACCGTT	1818.19	496	0.273	−1.299
TV	ACCGTA	1180.62	298	0.252	−1.377
TW	ACGTGG	606.25	837	1.381	0.323
TW	ACCTGG	1845.52	2403	1.302	0.264
TW	ACATGG	1494.02	1089	0.729	−0.316
TW	ACTTGG	1321.21	938	0.710	−0.343
TY	ACCTAC	2130.11	3648	1.713	0.538
TY	ACCTAT	1730.88	1778	1.027	0.027
TY	ACTTAC	1524.94	1383	0.907	−0.098
TY	ACGTAC	699.73	621	0.887	−0.119
TY	ACATAT	1401.21	1136	0.811	−0.210
TY	ACTTAT	1239.13	907	0.732	−0.312
TY	ACGTAT	568.59	408	0.718	−0.332
TY	ACATAC	1724.41	1138	0.660	−0.416
VA	GTGGCC	6082.92	9316	1.532	0.426
VA	GTAGCA	897.78	1347	1.500	0.406
VA	GTTGCT	1579.41	2217	1.404	0.339
VA	GTAGCT	1025.57	1407	1.372	0.316
VA	GTGGCT	4000.44	5252	1.313	0.272
VA	GTGGCG	1644.71	2099	1.276	0.244
VA	GTTGCA	1382.62	1728	1.250	0.223
VA	GTGGCA	3501.98	3859	1.102	0.097
VA	GTAGCC	1559.44	1363	0.874	−0.135
VA	GTTGCC	2401.60	1808	0.753	−0.284
VA	GTAGCG	421.64	216	0.512	−0.669
VA	GTTGCG	649.35	234	0.360	−1.021
VA	GTCGCG	831.37	284	0.342	−1.074
VA	GTCGCC	3074.82	992	0.323	−1.131
VA	GTCGCT	2022.16	406	0.201	−1.606
VA	GTCGCA	1770.19	318	0.180	−1.717
VC	GTCTGC	1410.66	2160	1.531	0.426
VC	GTCTGT	1188.18	1572	1.323	0.280
VC	GTTTGT	928.03	942	1.015	0.015
VC	GTATGT	602.60	594	0.986	−0.014
VC	GTGTGC	2790.71	2583	0.926	−0.077
VC	GTGTGT	2350.57	1996	0.849	−0.164
VC	GTTTGC	1101.80	830	0.753	−0.283
VC	GTATGC	715.44	411	0.574	−0.554
VD	GTAGAT	1225.65	1924	1.570	0.451
VD	GTGGAC	5400.58	7734	1.432	0.359
VD	GTTGAT	1887.55	2389	1.266	0.236
VD	GTGGAT	4780.91	5727	1.198	0.181
VD	GTAGAC	1384.52	1346	0.972	−0.028
VD	GTTGAC	2132.21	1791	0.840	−0.174
VD	GTCGAC	2729.91	602	0.221	−1.512
VD	GTCGAT	2416.67	445	0.184	−1.692
VE	GTAGAA	1456.83	2855	1.960	0.673
VE	GTGGAG	7599.48	11579	1.524	0.421
VE	GTTGAA	2243.56	2905	1.295	0.258
VE	GTGGAA	5682.64	6229	1.096	0.092
VE	GTAGAG	1948.24	2002	1.028	0.027
VE	GTTGAG	3000.36	1987	0.662	−0.412
VE	GTCGAG	3841.42	721	0.188	−1.673
VE	GTCGAA	2872.48	367	0.128	−2.058
VF	GTCTTC	2309.08	4216	1.826	0.602
VF	GTATTT	1023.16	1512	1.478	0.391
VF	GTCTTT	2017.40	2238	1.109	0.104
VF	GTTTTT	1575.70	1706	1.083	0.079
VF	GTTTTC	1803.52	1604	0.889	−0.117
VF	GTGTTT	3991.02	3257	0.816	−0.203
VF	GTGTTC	4568.05	3205	0.702	−0.354
VF	GTATTC	1171.09	721	0.616	−0.485
VG	GTTGGT	779.74	1617	2.074	0.729
VG	GTTGGA	1204.37	2315	1.922	0.653
VG	GTGGGC	4136.07	5977	1.445	0.368
VG	GTAGGA	782.04	1089	1.393	0.331
VG	GTTGGG	1175.77	1510	1.284	0.250
VG	GTTGGC	1632.96	1794	1.099	0.094
VG	GTAGGT	506.31	554	1.094	0.090
VG	GTGGGG	2978.07	3255	1.093	0.089
VG	GTGGGT	1974.96	2009	1.017	0.017
VG	GTAGGG	763.47	683	0.895	−0.111
VG	GTGGGA	3050.51	2599	0.852	−0.160
VG	GTAGGC	1060.34	676	0.638	−0.450
VG	GTCGGG	1505.36	734	0.488	−0.718
VG	GTCGGC	2090.72	734	0.351	−1.047
VG	GTCGGT	998.31	292	0.292	−1.229
VG	GTCGGA	1541.98	343	0.222	−1.503
VH	GTTCAT	911.79	1418	1.555	0.442
VH	GTACAT	592.06	773	1.306	0.267
VH	GTCCAC	1609.82	2085	1.295	0.259
VH	GTCCAT	1167.39	1313	1.125	0.118
VH	GTTCAC	1257.35	1319	1.049	0.048
VH	GTGCAC	3184.70	2856	0.897	−0.109
VH	GTACAC	816.44	613	0.751	−0.287
VH	GTGCAT	2309.44	1472	0.637	−0.450
VI	GTCATC	2367.78	5207	2.199	0.788
VI	GTCATT	1872.41	2827	1.510	0.412
VI	GTAATA	436.74	614	1.406	0.341
VI	GTAATT	949.63	1074	1.131	0.123
VI	GTTATT	1462.46	1595	1.091	0.087
VI	GTCATA	861.15	904	1.050	0.049
VI	GTTATA	672.60	702	1.044	0.043
VI	GTGATT	3704.20	2742	0.740	−0.301
VI	GTGATC	4684.19	3353	0.716	−0.334
VI	GTGATA	1703.61	1117	0.656	−0.422
VI	GTTATC	1849.37	1053	0.569	−0.563
VI	GTAATC	1200.86	577	0.480	−0.733
VK	GTAAAA	1288.46	1945	1.510	0.412
VK	GTCAAG	3290.24	3982	1.210	0.191
VK	GTGAAG	6509.08	7513	1.154	0.143
VK	GTAAAG	1668.70	1704	1.021	0.021
VK	GTCAAA	2540.51	2376	0.935	−0.067
VK	GTTAAA	1984.27	1777	0.896	−0.110
VK	GTGAAA	5025.89	4409	0.877	−0.131
VK	GTTAAG	2569.85	1171	0.456	−0.786
VL	GTTTTA	668.83	1311	1.960	0.673
VL	GTTCTT	1146.70	1859	1.621	0.483
VL	GTTTTG	1123.58	1737	1.546	0.436
VL	GTATTA	434.30	646	1.487	0.397
VL	GTCCTC	2129.16	3019	1.418	0.349
VL	GTTCTA	618.41	832	1.345	0.297
VL	GTCCTG	4424.65	5574	1.260	0.231
VL	GTCCTT	1468.14	1722	1.173	0.159
VL	GTGCTG	8753.31	10107	1.155	0.144
VL	GTCTTG	1438.54	1628	1.132	0.124
VL	GTACTA	401.55	447	1.113	0.107
VL	GTCCTA	791.76	874	1.104	0.099
VL	GTCTTA	856.32	863	1.008	0.008
VL	GTATTG	729.58	711	0.975	−0.026
VL	GTACTT	744.59	693	0.931	−0.072
VL	GTTCTC	1662.99	1501	0.903	−0.102
VL	GTGCTC	4212.12	3765	0.894	−0.112
VL	GTGCTA	1566.34	1286	0.821	−0.197
VL	GTTCTG	3455.90	2350	0.680	−0.386
VL	GTGTTG	2845.87	1910	0.671	−0.399
VL	GTGCTT	2904.43	1933	0.666	−0.407
VL	GTGTTA	1694.06	965	0.570	−0.563
VL	GTACTC	1079.84	541	0.501	−0.691
VL	GTACTG	2244.04	1121	0.500	−0.694
VM	GTCATG	2149.52	3308	1.539	0.431
VM	GTGATG	4252.41	3872	0.911	−0.094
VM	GTAATG	1090.17	935	0.858	−0.154
VM	GTTATG	1678.90	1056	0.629	−0.464
VN	GTCAAC	2052.00	3311	1.614	0.478
VN	GTAAAT	944.92	1518	1.606	0.474
VN	GTCAAT	1863.13	2155	1.157	0.146
VN	GTTAAT	1455.20	1325	0.911	−0.094
VN	GTGAAC	4059.49	3551	0.875	−0.134
VN	GTGAAT	3685.83	3110	0.844	−0.170
VN	GTAAAC	1040.71	854	0.821	−0.198
VN	GTTAAC	1602.73	880	0.549	−0.600
VP	GTTCCT	1434.04	2257	1.574	0.454
VP	GTTCCA	1384.03	1911	1.381	0.323
VP	GTGCCC	4055.45	4998	1.232	0.209
VP	GTACCT	931.17	1048	1.125	0.118
VP	GTCCCC	2049.96	2260	1.102	0.098
VP	GTCCCT	1836.02	2014	1.097	0.093
VP	GTACCA	898.70	963	1.072	0.069
VP	GTCCCG	742.77	786	1.058	0.057
VP	GTTCCC	1601.13	1506	0.941	−0.061
VP	GTCCCA	1772.00	1596	0.901	−0.105
VP	GTGCCT	3632.21	3062	0.843	−0.171
VP	GTGCCG	1469.43	1228	0.836	−0.179
VP	GTACCC	1039.67	809	0.778	−0.251
VP	GTGCCA	3505.55	2431	0.693	−0.366
VP	GTTCCG	580.15	279	0.481	−0.732
VP	GTACCG	376.71	161	0.427	−0.850
VQ	GTACAA	633.37	1049	1.656	0.505
VQ	GTTCAA	975.42	1485	1.522	0.420
VQ	GTCCAG	3487.32	3907	1.120	0.114
VQ	GTACAG	1768.65	1752	0.991	−0.009
VQ	GTTCAG	2723.79	2689	0.987	−0.013
VQ	GTGCAG	6898.98	6734	0.976	−0.024
VQ	GTCCAA	1248.85	1067	0.854	−0.157
VQ	GTGCAA	2470.60	1524	0.617	−0.483
VR	GTTCGA	463.33	867	1.871	0.627
VR	GTTCGT	334.59	580	1.733	0.550
VR	GTCCGA	593.21	805	1.357	0.305
VR	GTCCGC	1000.57	1332	1.331	0.286
VR	GTGCGC	1979.43	2543	1.285	0.251
VR	GTCCGT	428.38	549	1.282	0.248
VR	GTCCGG	1094.32	1346	1.230	0.207
VR	GTACGA	300.86	361	1.200	0.182
VR	GTAAGA	559.73	660	1.179	0.165
VR	GTGCGG	2164.91	2552	1.179	0.164
VR	GTCAGA	1103.65	1291	1.170	0.157
VR	GTACGT	217.26	253	1.165	0.152
VR	GTCAGG	1083.48	1238	1.143	0.133
VR	GTGAGG	2143.46	1986	0.927	−0.076
VR	GTGCGT	847.46	761	0.898	−0.108
VR	GTAAGG	549.51	444	0.808	−0.213
VR	GTTCGG	854.73	650	0.760	−0.274
VR	GTGCGA	1173.55	826	0.704	−0.351
VR	GTTCGC	781.50	545	0.697	−0.360
VR	GTGAGA	2183.35	1511	0.692	−0.368
VR	GTACGG	555.00	377	0.679	−0.387
VR	GTTAGA	862.01	556	0.645	−0.438
VR	GTACGC	507.46	286	0.564	−0.573
VR	GTTAGG	846.26	309	0.365	−1.007
VS	GTTTCT	1206.81	2161	1.791	0.583
VS	GTCTCC	1776.18	2936	1.653	0.503
VS	GTCAGC	2012.32	3223	1.602	0.471
VS	GTTTCA	975.63	1465	1.502	0.407
VS	GTCAGT	1269.59	1841	1.450	0.372
VS	GTATCT	783.62	1093	1.395	0.333
VS	GTATCA	633.51	806	1.272	0.241
VS	GTCTCT	1545.10	1847	1.195	0.178
VS	GTTTCC	1387.29	1604	1.156	0.145
VS	GTCTCG	469.69	542	1.154	0.143
VS	GTCTCA	1249.12	1333	1.067	0.065
VS	GTGTCC	3513.81	3722	1.059	0.058
VS	GTGTCG	929.19	860	0.926	−0.077
VS	GTGTCT	3056.67	2784	0.911	−0.093
VS	GTATCC	900.82	763	0.847	−0.166
VS	GTAAGT	643.89	499	0.775	−0.255
VS	GTGAGC	3980.98	2901	0.729	−0.316
VS	GTGTCA	2471.14	1710	0.692	−0.368
VS	GTTAGT	991.62	640	0.645	−0.438
VS	GTATCG	238.21	138	0.579	−0.546
VS	GTTTCG	366.85	202	0.551	−0.597
VS	GTGAGT	2511.63	1371	0.546	−0.605
VS	GTAAGC	1020.58	514	0.504	−0.686
VS	GTTAGC	1571.73	551	0.351	−1.048
VT	GTCACC	2294.69	4477	1.951	0.668
VT	GTCACT	1642.76	2452	1.493	0.401
VT	GTCACG	753.80	997	1.323	0.280
VT	GTAACT	833.15	1046	1.255	0.228
VT	GTCACA	1857.64	2207	1.188	0.172
VT	GTAACA	942.13	1096	1.163	0.151
VT	GTTACT	1283.09	1208	0.941	−0.060
VT	GTGACC	4539.59	4223	0.930	−0.072
VT	GTGACG	1491.24	1318	0.884	−0.123
VT	GTGACT	3249.88	2758	0.849	−0.164
VT	GTGACA	3674.98	2947	0.802	−0.221
VT	GTTACA	1450.92	1111	0.766	−0.267
VT	GTAACC	1163.79	758	0.651	−0.429
VT	GTTACC	1792.28	969	0.541	−0.615
VT	GTAACG	382.30	191	0.500	−0.694
VT	GTTACG	588.76	183	0.311	−1.169
VV	GTTGTA	655.54	1109	1.692	0.526
VV	GTTGTT	1009.55	1701	1.685	0.522
VV	GTAGTA	425.66	698	1.640	0.495
VV	GTGGTG	6476.64	9025	1.393	0.332
VV	GTGGTC	3273.84	4256	1.300	0.262
VV	GTAGTT	655.54	800	1.220	0.199
VV	GTTGTC	1292.55	1561	1.208	0.189
VV	GTGGTA	1660.38	1777	1.070	0.068
VV	GTGGTT	2557.05	2613	1.022	0.022
VV	GTTGTG	2557.05	2261	0.884	−0.123
VV	GTAGTG	1660.38	1161	0.699	−0.358
VV	GTAGTC	839.30	553	0.659	−0.417
VV	GTCGTC	1654.87	858	0.518	−0.657
VV	GTCGTG	3273.84	1250	0.382	−0.963
VV	GTCGTA	839.30	213	0.254	−1.371
VV	GTCGTT	1292.55	288	0.223	−1.501
VW	GTCTGG	1316.29	1763	1.339	0.292
VW	GTGTGG	2604.03	2451	0.941	−0.061
VW	GTATGG	667.58	578	0.866	−0.144
VW	GTTTGG	1028.10	824	0.801	−0.221
VY	GTCTAC	1602.79	2490	1.554	0.441
VY	GTTTAT	1017.23	1438	1.414	0.346
VY	GTATAT	660.53	875	1.325	0.281
VY	GTCTAT	1302.39	1544	1.186	0.170
VY	GTGTAC	3170.80	2654	0.837	−0.178
VY	GTTTAC	1251.87	1008	0.805	−0.217
VY	GTATAC	812.88	582	0.716	−0.334
VY	GTGTAT	2576.51	1804	0.700	−0.356
WA	TGGGCA	1469.77	1535	1.044	0.043
WA	TGGGCG	690.28	695	1.007	0.007
WA	TGGGCT	1678.97	1664	0.991	−0.009
WA	TGGGCC	2552.98	2498	0.978	−0.022
WC	TGGTGC	1057.38	1066	1.008	0.008
WC	TGGTGT	890.62	882	0.990	−0.010
WD	TGGGAC	2699.37	2807	1.040	0.039
WD	TGGGAT	2389.63	2282	0.955	−0.046
WE	TGGGAG	3580.00	3650	1.020	0.019
WE	TGGGAA	2677.00	2607	0.974	−0.026
WF	TGGTTT	1639.95	1735	1.058	0.056
WF	TGGTTC	1877.05	1782	0.949	−0.052
WG	TGGGGT	955.95	1064	1.113	0.107
WG	TGGGGC	2002.00	2179	1.088	0.085
WG	TGGGGA	1476.56	1454	0.985	−0.015
WG	TGGGGG	1441.49	1179	0.818	−0.201
WH	TGGCAT	971.42	1000	1.029	0.029
WH	TGGCAC	1339.58	1311	0.979	−0.022
WI	TGGATT	1537.91	1627	1.058	0.056
WI	TGGATA	707.30	714	1.009	0.009
WI	TGGATC	1944.78	1849	0.951	−0.051
WK	TGGAAG	3491.83	3645	1.044	0.043
WK	TGGAAA	2696.17	2543	0.943	−0.058
WL	TGGCTA	683.88	798	1.167	0.154
WL	TGGCTG	3821.78	4228	1.106	0.101
WL	TGGCTT	1268.11	1334	1.052	0.051
WL	TGGCTC	1839.05	1879	1.022	0.021
WL	TGGTTG	1242.54	855	0.688	−0.374
WL	TGGTTA	739.64	501	0.677	−0.390
WM	TGGATG	2335.00	2335	1.000	0.000
WN	TGGAAT	1978.70	2005	1.013	0.013
WN	TGGAAC	2179.30	2153	0.988	−0.012
WP	TGGCCC	1302.21	1381	1.061	0.059
WP	TGGCCG	471.84	486	1.030	0.030
WP	TGGCCA	1125.64	1123	0.998	−0.002
WP	TGGCCT	1166.31	1076	0.923	−0.081
WQ	TGGCAG	2983.56	2997	1.005	0.004
WQ	TGGCAA	1068.44	1055	0.987	−0.013
WR	TGGAGG	1198.99	1665	1.389	0.328
WR	TGGAGA	1221.30	1472	1.205	0.187
WR	TGGCGG	1210.98	979	0.808	−0.213
WR	TGGCGC	1107.23	895	0.808	−0.213
WR	TGGCGT	474.05	377	0.795	−0.229
WR	TGGCGA	656.45	481	0.733	−0.311
WS	TGGAGT	1031.75	1239	1.201	0.183
WS	TGGAGC	1635.35	1956	1.196	0.179
WS	TGGTCA	1015.12	898	0.885	−0.123
WS	TGGTCC	1443.44	1271	0.881	−0.127
WS	TGGTCT	1255.65	1076	0.857	−0.154
WS	TGGTCG	381.70	323	0.846	−0.167
WT	TGGACG	598.07	674	1.127	0.120
WT	TGGACA	1473.88	1559	1.058	0.056
WT	TGGACT	1303.39	1240	0.951	−0.050
WT	TGGACC	1820.65	1723	0.946	−0.055
WV	TGGGTC	1318.64	1378	1.045	0.044
WV	TGGGTG	2608.66	2633	1.009	0.009
WV	TGGGTA	668.77	665	0.994	−0.006
WV	TGGGTT	1029.93	950	0.922	−0.081
WW	TGGTGG	1559.00	1559	1.000	0.000
WY	TGGTAC	1444.91	1520	1.052	0.051
WY	TGGTAT	1174.09	1099	0.936	−0.066
YA	TATGCA	1120.39	2249	2.007	0.697
YA	TATGCT	1279.86	2296	1.794	0.584
YA	TATGCC	1946.11	2862	1.471	0.386
YA	TACGCG	647.56	622	0.961	−0.040
YA	TATGCG	526.19	482	0.916	−0.088
YA	TACGCC	2395.00	1402	0.585	−0.535
YA	TACGCA	1378.81	512	0.371	−0.991
YA	TACGCT	1575.07	444	0.282	−1.266
YC	TACTGC	1588.07	2411	1.518	0.418
YC	TACTGT	1337.61	1587	1.186	0.171
YC	TATTGT	1086.90	659	0.606	−0.500
YC	TATTGC	1290.42	646	0.501	−0.692
YD	TATGAT	2091.17	3707	1.773	0.572
YD	TATGAC	2362.22	3731	1.579	0.457
YD	TACGAC	2907.08	1653	0.569	−0.565
YD	TACGAT	2573.52	843	0.328	−1.116
YE	TATGAA	2515.85	5225	2.077	0.731
YE	TATGAG	3364.48	4722	1.403	0.339
YE	TACGAG	4140.53	2309	0.558	−0.584
YE	TACGAA	3096.14	861	0.278	−1.280
YF	TACTTC	2766.63	3380	1.222	0.200
YF	TATTTT	1964.12	2124	1.081	0.078
YF	TACTTT	2417.16	2201	0.911	−0.094
YF	TATTTC	2248.09	1691	0.752	−0.285
YG	TATGGA	1472.35	2874	1.952	0.669
YG	TATGGT	953.23	1665	1.747	0.558
YG	TATGGG	1437.38	2129	1.481	0.393
YG	TATGGC	1996.30	2749	1.377	0.320
YG	TACGGG	1768.93	1088	0.615	−0.486
YG	TACGGC	2456.76	1484	0.604	−0.504
YG	TACGGT	1173.10	448	0.382	−0.963
YG	TACGGA	1811.96	633	0.349	−1.052
YH	TACCAC	1862.81	2378	1.277	0.244
YH	TACCAT	1350.85	1420	1.051	0.050
YH	TATCAT	1097.67	1021	0.930	−0.072
YH	TATCAC	1513.67	1006	0.665	−0.409
YI	TACATC	2684.66	3935	1.466	0.382
YI	TACATT	2122.99	2162	1.018	0.018
YI	TATATT	1725.09	1554	0.901	−0.104
YI	TACATA	976.39	846	0.866	−0.143
YI	TATATA	793.39	648	0.817	−0.202
YI	TATATC	2181.48	1339	0.614	−0.488
YK	TACAAG	3508.58	4372	1.246	0.220
YK	TACAAA	2709.10	2847	1.051	0.050
YK	TATAAA	2201.34	2262	1.028	0.027
YK	TATAAG	2850.98	1789	0.628	−0.466
YL	TACCTG	4522.42	6324	1.398	0.335
YL	TATTTA	711.20	966	1.358	0.306
YL	TACCTC	2176.20	2598	1.194	0.177
YL	TACTTG	1470.33	1701	1.157	0.146
YL	TATTTG	1194.75	1358	1.137	0.128
YL	TACCTA	809.25	876	1.082	0.079
YL	TACCTT	1500.58	1449	0.966	−0.035
YL	TATCTT	1219.33	1166	0.956	−0.045
YL	TACTTA	875.24	763	0.872	−0.137
YL	TATCTA	657.58	541	0.823	−0.195
YL	TATCTC	1768.32	1087	0.615	−0.487
YL	TATCTG	3674.80	1751	0.476	−0.741
YM	TACATG	2325.97	3055	1.313	0.273
YM	TATATG	1890.03	1161	0.614	−0.487
YN	TACAAC	2442.24	3341	1.368	0.313
YN	TACAAT	2217.44	2200	0.992	−0.008
YN	TATAAT	1801.83	1629	0.904	−0.101
YN	TATAAC	1984.50	1276	0.643	−0.442
YP	TACCCG	668.65	1004	1.502	0.406
YP	TACCCA	1595.15	1925	1.207	0.188
YP	TATCCA	1296.18	1438	1.109	0.104
YP	TACCCC	1845.38	1961	1.063	0.061
YP	TATCCT	1343.02	1379	1.027	0.026
YP	TACCCT	1652.79	1558	0.943	−0.059
YP	TATCCC	1499.51	937	0.625	−0.470
YP	TATCCG	543.32	242	0.445	−0.809
YQ	TACCAG	3987.12	5013	1.257	0.229
YQ	TATCAA	1160.22	1179	1.016	0.016
YQ	TACCAA	1427.83	1397	0.978	−0.022
YQ	TATCAG	3239.83	2226	0.687	−0.375
YR	TACCGC	1307.70	2153	1.646	0.499
YR	TACCGA	775.30	990	1.277	0.244
YR	TACAGA	1442.41	1834	1.271	0.240
YR	TACCGG	1430.23	1796	1.256	0.228
YR	TACAGG	1416.06	1671	1.180	0.166
YR	TACCGT	559.87	642	1.147	0.137
YR	TATCGA	629.99	570	0.905	−0.100
YR	TATCGT	454.94	383	0.842	−0.172
YR	TATAGA	1172.07	827	0.706	−0.349
YR	TATCGG	1162.17	629	0.541	−0.614
YR	TATAGG	1150.66	560	0.487	−0.720
YR	TATCGC	1062.60	509	0.479	−0.736
YS	TACAGC	2204.13	3590	1.629	0.488
YS	TACTCG	514.46	783	1.522	0.420
YS	TACAGT	1390.60	1887	1.357	0.305
YS	TATTCA	1111.75	1210	1.088	0.085
YS	TACTCC	1945.47	2088	1.073	0.071
YS	TATTCT	1375.18	1466	1.066	0.064
YS	TACTCA	1368.18	1188	0.868	−0.141
YS	TATTCC	1580.84	1306	0.826	−0.191
YS	TACTCT	1692.37	1173	0.693	−0.367
YS	TATAGT	1129.96	728	0.644	−0.440
YS	TATTCG	418.04	229	0.548	−0.602
YS	TATAGC	1791.02	874	0.488	−0.717
YT	TACACG	697.26	1311	1.880	0.631
YT	TACACC	2122.58	2696	1.270	0.239
YT	TACACA	1718.31	2158	1.256	0.228
YT	TACACT	1519.54	1409	0.927	−0.076
YT	TATACT	1234.74	1049	0.850	−0.163
YT	TATACA	1396.25	1049	0.751	−0.286
YT	TATACC	1724.75	1063	0.616	−0.484
YT	TATACG	566.57	245	0.432	−0.838
YV	TATGTT	986.79	1723	1.746	0.557
YV	TATGTA	640.76	1113	1.737	0.552
YV	TATGTC	1263.40	1862	1.474	0.388
YV	TATGTG	2499.39	3382	1.353	0.302
YV	TACGTG	3075.90	2279	0.741	−0.300
YV	TACGTC	1554.82	991	0.637	−0.450
YV	TACGTA	788.55	284	0.360	−1.021
YV	TACGTT	1214.40	390	0.321	−1.136
YW	TACTGG	1609.87	2212	1.374	0.318
YW	TATTGG	1308.13	706	0.540	−0.617
YY	TACTAC	2256.03	2854	1.265	0.235
YY	TATTAT	1489.60	1459	0.979	−0.021
YY	TACTAT	1833.19	1760	0.960	−0.041
YY	TATTAC	1833.19	1339	0.730	−0.314

Claims

1. A method of making a modified viral genome comprising: a. obtaining the nucleotide sequence of a parent protein encoding sequence of a parent virusb. rearranging synonymous codons from the protein encoding sequence of the parent virus to obtain a modified protein encoding sequence, wherein said rearrangement provides a reduced codon pair bias relative to a mammalian host over the modified protein encoding sequence, in comparison to the encoding region of the parent virus, without changing codon usage of the parent virus, wherein the codon pair bias is calculated by the following formula:

2. The method of claim 1, in which rearranging the synonymous codons comprises the step of randomly selecting and exchanging pairs of codons encoding the same amino acid and determining whether codon pair bias is reduced by the exchange.

3. The method of claim 2, wherein the step is repeated until the codon pair bias is reduced by a desired amount.

4. The method of claim 2, wherein the step is repeated until the codon pair bias converges on or near an optimal value.

5. The method of claim 1, wherein steps (a) and (b) are implemented on a computer.

6. The method of claim 1, wherein step (c) is achieved by de novo synthesis of the modified protein-encoding sequence.

7. The method of claim 6, wherein the entire genome is substituted with the synthesized DNA.

8. The method of claim 6, wherein a portion of the viral genome is substituted with the synthesized DNA.

9. The method of claim 1, wherein the parent virus is a natural isolate.

10. The method of claim 1, wherein the parent virus is a mutant of a natural isolate.

11. The method of claim 1, wherein the modified protein encoding sequence has a codon pair bias at least 0.1 less than the codon pair bias of the parent protein encoding sequence.

12. The method of claim 1, wherein the modified protein encoding sequence has a codon pair bias at least 0.2 less than the codon pair bias of the parent protein encoding sequence.

13. The method of claim 1, wherein the modified protein encoding sequence has a codon pair bias at least 0.3 less than the codon pair bias of the parent protein encoding sequence.

14. The method of claim 1, wherein the modified protein encoding sequence has a codon pair bias at least 0.4 less than the codon pair bias of the parent protein encoding sequence.

15. The method of claim 1, wherein the modified protein encoding sequence has a codon pair bias of −0.05 or less.

16. The method of claim 1, wherein the modified protein encoding sequence has a codon pair bias of −0.1 or less.

17. The method of claim 1, wherein the modified protein encoding sequence has a codon pair bias of −0.3 or less.

18. The method of claim 1, wherein the modified protein encoding sequence has a codon pair bias of −0.4 or less.

19. The method of claim 1, wherein the modified protein encoding sequence is modified over a length of at least 100 nucleotides.

20. The method of claim 1, wherein the modified protein encoding sequence is modified over a length of at least 500 nucleotides.

21. The method of claim 1, wherein the modified protein encoding sequence is modified over a length of at least 1000 nucleotides.

22. The method of claim 1, wherein the parent virus is a poliovirus, rhinovirus, influenza virus, severe acute respiratory syndrome (SARS) coronavirus, Human Immunodeficiency Virus (HIV), Hepatitis C Virus (HCV), infectious bronchitis virus, Ebolavirus, Marburg virus, dengue fever virus, West Nile disease virus, Epstein-Barr virus (EBV), yellow fever virus, Poxvirus, Herpes virus, Papillomavirus, or Adenovirus.

23. A method of making a modified virus comprising, making a modified viral genome according to the method of claim 1; andinserting the modified viral genome into a host cell, whereby a modified virus is produced.

24. The method of claim 1, wherein the parent virus is a DNA, RNA, double-stranded, or single-stranded virus.

25. The method of claim 1, wherein the modified viral genome is a DNA or an RNA nucleotide sequence.